Methods of increasing abiotic stress tolerance and/or biomass in plants

ABSTRACT

Provided are methods of increasing the tolerance of a plant to abiotic stresses and/or increasing the biomass and/or increasing the yield of a plant by expressing within the plant an exogenous polynucleotide encoding a polypeptide homologous to SEQ ID NO:240, such as the polynucleotide set forth by SEQ ID NO:14.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.13/874,500 filed on May 1, 2013, which is a continuation of U.S. patentapplication Ser. No. 12/457,199 filed on Jun. 3, 2009, now U.S. Pat. No.8,481,812, which is a continuation of U.S. patent application Ser. No.11/284,236 filed on Nov. 22, 2005, now U.S. Pat. No. 7,554,007, which isa Continuation-In-Part (CIP) of PCT Patent Application No.PCT/IL2004/000431 filed on May 20, 2004, which claims the benefit ofpriority of U.S. Provisional Patent Application No. 60/472,433 filed onMay 22, 2003.

U.S. patent application Ser. No. 11/284,236 also claims the benefit ofpriority of U.S. Provisional Patent Application No. 60/707,957 filed onAug. 15, 2005.

The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 65113SequenceListing.txt, created on Jan. 25,2016, comprising 425,492 bytes, submitted concurrently with the filingof this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to methods of increasing abiotic stresstolerance and/or biomass in plants and, more particularly, to plantsexpressing exogenous abiotic stress-tolerance genes.

Abiotic stress (also referred to as “environmental stress”) conditionssuch as salinity, drought, flood, suboptimal temperature and toxicchemical pollution, cause substantial damage to agricultural plants.Most plants have evolved strategies to protect themselves against theseconditions. However, if the severity and duration of the stressconditions are too great, the effects on plant development, growth andyield of most crop plants are profound. Furthermore, most crop plantsare very susceptible to abiotic stress (ABS) and thus necessitateoptimal growth conditions for commercial crop yields. Continuousexposure to stress causes major alterations in plant metabolism whichultimately lead to cell death and consequently yields losses. Thus,despite extensive research and the use of sophisticated and intensivecrop-protection measures, losses due to abiotic stress conditions remainin the billions of dollars annually (1,2).

Developing stress-tolerant plants is a strategy that has the potentialto solve or mediate at least some of these problems. However,traditional plant breeding strategies used to develop new lines ofplants that exhibit tolerance to ABS are relatively inefficient sincethey are tedious, time consuming and of unpredictable outcome.Furthermore, limited germplasm resources for stress tolerance andincompatibility in crosses between distantly related plant speciesrepresent significant problems encountered in conventional breeding.Additionally, the cellular processes leading to ABS tolerance arecomplex in nature and involve multiple mechanisms of cellular adaptationand numerous metabolic pathways (4-7).

Genetic engineering efforts, aimed at conferring abiotic stresstolerance to transgenic crops, have been described in the prior art.Studies by Apse and Blumwald (Curr Opin Biotechnol. 13:146-150, 2002),Quesada et al. (Plant Physiol. 130:951-963, 2002), Holmström et al.(Nature 379: 683-684, 1996), Xu et al. (Plant Physiol 110: 249-257,1996), Pilon-Smits and Ebskamp (Plant Physiol 107: 125-130, 1995) andTarczynski et al. (Science 259: 508-510, 1993) have all attempted atgenerating stress tolerant plants.

In addition, several U.S. patents and patent applications also describepolynucleotides associated with stress tolerance and their use ingenerating stress tolerant plants. U.S. Pat. Nos. 5,296,462 and5,356,816 describe transforming plants with polynucleotides encodingproteins involved in cold adaptation in Arabidopsis thaliana, to therebypromote cold tolerance in the transformed plants.

U.S. Pat. No. 6,670,528 describes transforming plants withpolynucleotides encoding polypeptides binding to stress responsiveelements, to thereby promote tolerance of the transformed plants toabiotic stress.

U.S. Pat. No. 6,720,477 describes transforming plants with apolynucleotide encoding a signal transduction stress-related protein,capable of increasing tolerance of the transformed plants to abioticstress.

U.S. application Ser. Nos. 09/938,842 and 10/342,224 describe abioticstress-related genes and their use to confer upon plants tolerance toabiotic stress.

U.S. application Ser. No. 10/231,035 describes overexpressing amolybdenum cofactor sulfurase in plants to thereby increase theirtolerance to abiotic stress.

Although the above described studies were at least partially successfulin generating stress tolerant plants, there remains a need for stresstolerant genes which can be utilized to generate plants tolerant of awide range of abiotic stress conditions.

While reducing the present invention to practice, the present inventorshave identified through bioinformatic and laboratory studies severalnovel abiotic stress-tolerance genes, which can be utilized to increasetolerance to abiotic stress and/or biomass in plants.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided amethod of increasing tolerance of a plant to an abiotic stress. Themethod includes expressing within the plant an exogenous polynucleotideat least 90% homologous to a polynucleotide selected from the groupconsisting of SEQ ID NOs: 1-18, 93-98 and 247-252.

According to an additional aspect of the present invention there isprovided a method of increasing tolerance of a plant to an abioticstress. The method includes expressing within the plant an exogenouspolynpeptide including an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 39-92 and 105-230.

According to another aspect of the present invention there is provided amethod of increasing biomass and/or yield of a plant. The methodincludes expressing within the plant an exogenous polynucleotide atleast 90% homologous to a polynucleotide selected from the groupconsisting of SEQ ID NOs: 1-18, 93-98 and 247-252.

According to still additional aspect of the present invention there isprovided a method of increasing biomass and/or yield of a plant. Themethod includes expressing within the plant an exogenous polypeptideincluding an amino acid sequence selected from the group consisting ofSEQ ID NOs: 39-92 and 105-230.

According to yet another aspect of the present invention there isprovided a plant cell comprising an exogenous polynucleotide at least90% homologous to a polynucleotide selected from the group consisting ofSEQ ID NOs: 1-18, 93-98 and 247-252.

According to yet another aspect of the present invention there isprovided a plant cell comprising an exogenous polynucleotide encoding apolypeptide including an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 39-92 and 105-230.

According to still another aspect of the present invention there isprovided a nucleic acid construct, including a polynucleotide at least90% homologous to a nucleotide sequence selected from the groupconsisting of SEQ ID NOs: 1-18, 93-98 and 247-252 and a promoter capableof directing transcription of the polynucleotide in a host cell.

According to another aspect of the present invention there is provided anucleic acid construct, including a polynucleotide encoding apolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 39-92 and 105-230 and a promoter capable ofdirecting transcription of the polynucleotide in a host cell.

According to further yet another aspect of the present invention thereis provided an isolated polypeptide, including an amino acid sequence atleast 90% homologous to the amino acid sequence encoded by apolynucleotide selected from the group consisting of SEQ ID NOs: 1-18,93-98 and 247-252.

According to an additional aspect of the present invention there isprovided an isolated polypeptide including an amino acid sequenceselected from the group consisting of SEQ ID NOs: 39-92 and 105-230.

According to further features in preferred embodiments of the inventiondescribed below, the expressing is effected by (i) transforming a cellof the plant with the exogenous polynucleotide; (ii) generating a matureplant from the cell; and (iii) cultivating the mature plant underconditions suitable for expressing the exogenous polynucleotide withinthe mature plant.

According to still further features in the described preferredembodiments the transforming is effected by introducing to the plantcell a nucleic acid construct including the exogenous polynucleotide andat least one promoter capable of directing transcription of theexogenous polynucleotide in the plant cell.

According to still further features in the described preferredembodiments the at least one promoter is a constitutive promoter.

According to still further features in the described preferredembodiments the constitutive promoter is CaMV 35S promoter.

According to still further features in the described preferredembodiments the constitutive promoter is At6669 promoter.

According to still further features in the described preferredembodiments the at least one promoter is an inducible promoter.

According to still further features in the described preferredembodiments the inducible promoter is an abiotic stress induciblepromoter.

According to still further features in the described preferredembodiments the at least one promoter is a tissue-specific promoter.

According to still further features in the described preferredembodiments the expressing is effected by infecting the plant with avirus including the exogenous polynucleotide.

According to still further features in the described preferredembodiments the virus is an avirulent virus.

According to still further features in the described preferredembodiments the abiotic stress is selected from the group consisting ofsalinity, water deprivation, low temperature, high temperature, heavymetal toxicity, anaerobiosis, nutrient deficiency, nutrient excess,atmospheric pollution and UV irradiation.

According to still further features in the described preferredembodiments the plant is a dicotyledonous plant.

According to still further features in the described preferredembodiments the plant is a monocotyledonous plant.

According to still further features in the described preferredembodiments the plant cell forms a part of a plant.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing methods of utilizing novelabiotic stress-tolerance genes to increase plants tolerance to abioticstress and/or biomass and/or commercial yield.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

FIG. 1 is a flow chart illustrating a process of identifying putativeplant stress-tolerance genes from nucleic-acid sequence databases;

FIG. 2 is a photograph of tomato seedling of a sensitive line (Evoline1, Evogene, Rehovot, Israel) as seen on the left and a tolerant line(Evoline 2, Evogene, Rehovot, Israel) as seen on the right following 4week growth under water irrigation containing 300 mM NaCl. Evoline1 wasproved for several seasons as being a relatively salt-sensitive line.Evoline 2 was proved for several seasons as being a highly salt-tolerantline;

FIGS. 3A-3D are photographs illustrating a T2 transgenic Arabidopsisthaliana mature plant at flowering stage, expressing exogenousluciferase transgene from the At6669 promoter. The same plant is shownin FIGS. 3A and 3B and a second plant is shown in FIGS. 3C and 3D. FIGS.3A and 3C are photographs taken under normal light conditions and FIGS.3B and 3D are photographs taken in the dark. Strong illuminationindicative of luciferase expression is observed in the flower and roottissues;

FIG. 4 is a bar graph illustrating the mean plant dry weight oftransgenic T₂ A. thaliana plants grown under salinity stress conditions(irrigated with 100 mM NaCl solution), as compared with similar plantsgrown under normal conditions (irrigated with water only). The plantswere transformed with putative stress tolerance genes of the presentinvention (ABST_1, 6, 19, 22, 27, 36, 37) and the effect of thepromoters (35S vs. 6669) on biomass was examined;

FIG. 5 illustrates the mean fresh weight of transgenic T₁ A. thalianaplants grown under normal and stress conditions (irrigated with 0 or 100M NaCl solution, respectively). The plants were transformed withputative stress tolerance genes, or with a luciferase reporter gene(control), positioned under the transcriptional control of the At6669promoter. Means followed by the same letter are not significantlydifferent according to a one way ANOVA T-Test;

FIG. 6A illustrates the mean fresh weight of T₂ A. thaliana plants grownunder normal or stress conditions (irrigated with 0 or 100 M NaClsolution, respectively). The plants were transformed with the putativestress tolerance genes of the present invention, or with luciferasereporter gene (control), positioned under the transcriptional control ofthe 35S promoter. Means followed by the same letter are notsignificantly different according to a one way ANOVA T-Test;

FIG. 6B illustrates the mean fresh weight of T₂ A. thaliana plants grownunder normal or stress conditions (irrigated with 0 or 100 M NaClsolution, respectively). The plants were transformed with the putativestress tolerance genes of the present invention, or with luciferasereporter gene (control), positioned under the transcriptional control ofthe At6669 promoter. Means followed by the same letter are notsignificantly different according to a one way ANOVA T-Test;

FIG. 7 illustrates the total seed weight from T₂ A. thaliana plantsover-expressing the ABST genes of the present invention regulated by the6669 promoter grown under regular conditions. Means followed by the sameletter are not significantly different according to a one way ANOVAT-Test;

FIGS. 8A-8D are photographs depicting control and transgenic tomatoplants (of the genetic background of Evoline3) of the present inventionillustrating the increase in yield following over-expression of theputative ABST genes of the present invention. FIG. 8A is a photograph ofa tomato plant over-expressing ABST_1, SEQ ID NO. 1 (right; 28) comparedto its isogenic line that does not carry the gene (left; 29). FIG. 8B isa photograph of roots from a tomato plant over-expressing ABST_1, SEQI.D. NO. 1 (right; 28) compared to its isogenic line that does not carrythe gene (left; 29). FIG. 8C is a photograph of tomato plant canopies ofa plant over-expressing ABST_36, SEQ I.D. NO. 13 (right; 30) compared toa control plant (left; 31). FIG. 8D is a photograph of total fruits of atomato plant over-expressing ABST_36, SEQ I.D. NO. 13 (right; 30)compared to a control plant (left; 31); and

FIGS. 9A-9C are line graphs illustrating the relative expression ofputative ABST genes in stress tolerant tomato leaves (Evoline 2) versusstress sensitive tomato leaves (Evoline 1). FIG. 9A illustrates therelative expression of ABST_36 gene in Evoline 2 tomato leaves followingsalt induction compared to its expression in leaves of the Evoline 1variety. FIG. 9B illustrates the relative expression of ABST_36 gene inEvoline 2 tomato roots following salt induction compared to itsexpression in roots of the Evoline 1 variety. FIG. 9C illustrates therelative expression of ABST_37 gene in Evoline 2 tomato leaves followingsalt induction compared to its expression in leaves of the Evoline 1variety.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention is of methods of increasing plants tolerance toabiotic stress and/or biomass by utilizing novel abiotic stresstolerance genes and of plants exhibiting increased tolerance to stressconditions and/or increased capacity to accumulate biomass.

The principles and operation of the present invention may be betterunderstood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details of construction and the arrangement of the components setforth in the following description or illustrated in the drawings. Theinvention is capable of other embodiments or of being practiced orcarried out in various ways. Also, it is to be understood that thephraseology and terminology employed herein is for the purpose ofdescription and should not be regarded as limiting.

While reducing the present invention to practice, the present inventorswhile employing bioinformatic techniques, identified polynucleotidesequences which encode putative abiotic-stress tolerance (ABST) proteins(Examples 1 and 11). Selected sequences were isolated (Examples 3 and12), cloned into expression vectors (Example 4 and 13) and introducedinto Arabidopsis thaliana plants (Example 8) and tomato plants (Example10). These plants, which were grown under salinity stress conditions, orunder normal conditions, exhibited significantly higher biomass ascompared with similar plants not carrying the exogenous ABST genes(Examples 9 and 10).

Thus, according to one aspect of the present invention, there isprovided a method of increasing tolerance of a plant to an abioticstress and/or plant biomass. The method includes expressing within theplant an exogenous polynucleotide at least 70% homologous, preferably atleast 80% homologous, more preferably at least 85% homologous, mostpreferably at least 90% homologous to a polynucleotide selected from thegroup consisting of SEQ ID NOs: 1-18, 93-98 and 247-252. Alternatively,the exogenous polynucleotide of the present invention encodes apolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NOs: 39-92 and 105-230.

As demonstrated in Example 8 herein below, introduction of SEQ ID NOs:1, 4, 9, 13 and 14 into Arabidopsis thaliana plants increased planttolerance to abiotic stress, such as a salinity stress as measured by anincrease in fresh weight, dry weight, and/or seed weight. Transgenictomato plants showed an increase in fresh canopy weight, dry weight,root weight, seed weight and increase in total fruit yield duringabiotic stress, such as salinity or drought stress followingintroduction of these polynucleotide sequences as demonstrated inExample 10.

The nucleic acid sequences of the present invention may be altered, tofurther improve expression levels for example, by optimizing the nucleicacid sequence in accordance with the preferred codon usage for aparticular plant cell type which is selected for the expression of thepolypeptides of the present invention.

Construction of synthetic genes by altering the codon usage is describedin for example PCT Patent Application WO 93/07278.

Alternatively, ortholog sequences in a particular plant may beidentified (e.g. by bioinformatics techniques) as described in Example10. Following qualification, these may be used to direct the expressionof the polypeptides of the present invention in a particular plantspecies. Since this may increase the probability of gene silencing, itmay be preferable to optimize the nucleic acid sequence in accordancewith the preferred codon usage as described above.

The phrase “abiotic stress” used herein refers to any adverse effect onmetabolism, growth, reproduction and/or viability of a plant.Accordingly, abiotic stress can be induced by suboptimal environmentalgrowth conditions such as, for example, salinity, water deprivation,flooding, low or high temperature, heavy metal toxicity, anaerobiosis,nutrient deficiency, atmospheric pollution or UV irradiation.

The phrase “abiotic stress tolerance” as used herein refers to theability of a plant to endure an abiotic stress without suffering asubstantial alteration in metabolism, growth, productivity and/orviability.

The polynucleotides of the present invention may enhance abiotic stresstolerance by any mechanism. The polynucleotides may enhance abioticstress tolerance by encoding for polypeptides which increase the amountof water available to the plant. For example, SEQ ID NO: 13 encodes apolypeptide that enhances symplastic water transport. Alternatively thepolynucleotides may enhance abiotic stress tolerance by encoding forpolypeptides which are involved in enhancing the expression of otherproteins involved in abiotic stress tolerance. For example, SEQ ID NO: 1encodes a cytoplasmic ribosomal protein and SEQ ID NO: 14 is atranscription factor.

A suitable plant for use with the method of the present invention can beany monocotyledonous or dicotyledonous plant including, but not limitedto, maize, wheat, barely, rye, oat, rice, soybean, peanut, pea, lentiland alfalfa, cotton, rapeseed, canola, pepper, sunflower, potato,tobacco, tomato, eggplant, eucalyptus, a tree, an ornamental plant, aperennial grass and a forage crop.

As used herein, the term “exogenous polynucleotide” refers to a nucleicacid sequence which is not naturally expressed within the plant butwhich, when introduced into the plant either in a stable or transientmanner, produces at least one polypeptide product.

Expressing the exogenous polynucleotide of the present invention withinthe plant can be effected by transforming one or more cells of the plantwith the exogenous polynucleotide, followed by generating a mature plantfrom the transformed cells and cultivating the mature plant underconditions suitable for expressing the exogenous polynucleotide withinthe mature plant.

Preferably, the transformation is effected by introducing to the plantcell a nucleic acid construct which includes the exogenouspolynucleotide of the present invention and at least one promotercapable of directing transcription of the exogenous polynucleotide inthe plant cell. Further details of suitable transformation approachesare provided hereinbelow.

As used herein, the term “promoter” refers to a region of DNA which liesupstream of the transcriptional initiation site of a gene to which RNApolymerase binds to initiate transcription of RNA. The promoter controlswhere (e.g., which portion of a plant, which organ within an animal,etc.) and/or when (e.g., which stage or condition in the lifetime of anorganism) the gene is expressed.

Any suitable promoter sequence can be used by the nucleic acid constructof the present invention. Preferably the promoter is a constitutivepromoter, a tissue-specific, or an abiotic stress-inducible promoter.

Suitable constitutive promoters include, for example, CaMV 35S promoter(SEQ ID NO: 19; Odell et al., Nature 313:810-812, 1985); ArabidopsisAt6669 promoter (SEQ ID NO: 20); maize Ubi 1 (Christensen et al., PlantSol. Biol. 18:675-689, 1992); rice actin (McElroy et al., Plant Cell2:163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81:581-588,1991); and Synthetic Super MAS (Ni et al., The Plant Journal 7: 661-76,1995). Other constitutive promoters include those in U.S. Pat. Nos.5,659,026, 5,608,149; 5.608,144; 5,604,121; 5,569,597: 5,466,785;5,399,680; 5,268,463; and 5,608,142.

Suitable tissue-specific promoters include, but not limited to,leaf-specific promoters such as described, for example, by Yamamoto etal., Plant J. 12:255-265, 1997; Kwon et al., Plant Physiol. 105:357-67,1994; Yamamoto et al., Plant Cell Physiol. 35:773-778, 1994; Gotor etal., Plant J. 3:509-18, 1993; Orozco et al., Plant Mol. Biol.23:1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA90:9586-9590, 1993.

Suitable abiotic stress-inducible promoters include, but not limited to,salt-inducible promoters such as RD29A (Yamaguchi-Shinozalei et al.,Mol. Gen. Genet. 236:331-340, 1993); drought-inducible promoters such asmaize rab17 gene promoter (Pla et. al., Plant Mol. Biol. 21:259-266,1993), maize rab28 gene promoter (Busk et. al., Plant J. 11:1285-1295,1997) and maize Ivr2 gene promoter (Pelleschi et. al., Plant Mol. Biol.39:373-380, 1999); and heat-inducible promoters such as heat tomatohsp80-promoter from tomato (U.S. Pat. No. 5,187,267).

The nucleic acid construct of the present invention preferably furtherincludes an appropriate selectable marker and/or an origin ofreplication. Preferably, the nucleic acid construct utilized is ashuttle vector, which can propagate both in E. coli (wherein theconstruct comprises an appropriate selectable marker and origin ofreplication) and be compatible for propagation in cells. The constructaccording to the present invention can be, for example, a plasmid, abacmid, a phagemid, a cosmid, a phage, a virus or an artificialchromosome.

The nucleic acid construct of the present invention can be utilized tostably or transiently transform plant cells. In stable transformation,the exogenous polynucleotide of the present invention is integrated intothe plant genome and as such it represents a stable and inherited trait.In transient transformation, the exogenous polynucleotide is expressedby the cell transformed but it is not integrated into the genome and assuch it represents a transient trait.

There are various methods of introducing foreign genes into bothmonocotyledonous and dicotyledonous plants (Potrykus, I., Annu. Rev.Plant. Physiol., Plant. Mol. Biol. (1991) 42:205-225; Shimamoto et al.,Nature (1989) 338:274-276).

The principle methods of causing stable integration of exogenous DNAinto plant genomic DNA include two main approaches:

(i) Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev.Plant Physiol. 38:467-486; Klee and Rogers in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes, eds. Schell, J., and Vasil, L. K., Academic Publishers, SanDiego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds.Kung, S. and Arntzen, C. J., Butterworth Publishers, Boston, Mass.(1989) p. 93-112.

(ii) Direct DNA uptake: Paszkowski et al., in Cell Culture and SomaticCell Genetics of Plants, Vol. 6, Molecular Biology of Plant NuclearGenes eds. Schell, J., and Vasil, L. K., Academic Publishers, San Diego,Calif. (1989) p. 52-68; including methods for direct uptake of DNA intoprotoplasts, Toriyama, K. et al. (1988) Bio/Technology 6:1072-1074. DNAuptake induced by brief electric shock of plant cells: Zhang et al.Plant Cell Rep. (1988) 7:379-384. Fromm et al. Nature (1986)319:791-793. DNA injection into plant cells or tissues by particlebombardment, Klein et al. Bio/Technology (1988) 6:559-563; McCabe et al.Bio/Technology (1988) 6:923-926; Sanford, Physiol. Plant. (1990)79:206-209; by the use of micropipette systems: Neuhaus et al., Theor.Appl. Genet. (1987) 75:30-36; Neuhaus and Spangenberg, Physiol. Plant.(1990) 79:213-217; glass fibers or silicon carbide whiskertransformation of cell cultures, embryos or callus tissue, U.S. Pat. No.5,464,765 or by the direct incubation of DNA with germinating pollen,DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman,G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p.197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83:715-719.

The Agrobacterium system includes the use of plasmid vectors thatcontain defined DNA segments that integrate into the plant genomic DNA.Methods of inoculation of the plant tissue vary depending upon the plantspecies and the Agrobacterium delivery system. A widely used approach isthe leaf disc procedure which can be performed with any tissue explantthat provides a good source for initiation of whole plantdifferentiation. Horsch et al. in Plant Molecular Biology Manual A5,Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementaryapproach employs the Agrobacterium delivery system in combination withvacuum infiltration. The Agrobacterium system is especially viable inthe creation of transgenic dicotyledonous plants.

There are various methods of direct DNA transfer into plant cells. Inelectroporation, the protoplasts are briefly exposed to a strongelectric field. In microinjection, the DNA is mechanically injecteddirectly into the cells using very small micropipettes. In microparticlebombardment, the DNA is adsorbed on microprojectiles such as magnesiumsulfate crystals or tungsten particles, and the microprojectiles arephysically accelerated into cells or plant tissues.

Following stable transformation plant propagation is exercised. The mostcommon method of plant propagation is by seed. Regeneration by seedpropagation, however, has the deficiency that due to heterozygositythere is a lack of uniformity in the crop, since seeds are produced byplants according to the genetic variances governed by Mendelian rules.Basically, each seed is genetically different and each will grow withits own specific traits. Therefore, it is preferred that the transformedplant be produced such that the regenerated plant has the identicaltraits and characteristics of the parent transgenic plant. Therefore, itis preferred that the transformed plant be regenerated bymicropropagation which provides a rapid, consistent reproduction of thetransformed plants.

Micropropagation is a process of growing new generation plants from asingle piece of tissue that has been excised from a selected parentplant or cultivar. This process permits the mass reproduction of plantshaving the preferred tissue expressing the fusion protein. The newgeneration plants which are produced are genetically identical to, andhave all of the characteristics of, the original plant. Micropropagationallows mass production of quality plant material in a short period oftime and offers a rapid multiplication of selected cultivars in thepreservation of the characteristics of the original transgenic ortransformed plant. The advantages of cloning plants are the speed ofplant multiplication and the quality and uniformity of plants produced.

Micropropagation is a multi-stage procedure that requires alteration ofculture medium or growth conditions between stages. Thus, themicropropagation process involves four basic stages: Stage one, initialtissue culturing; stage two, tissue culture multiplication; stage three,differentiation and plant formation; and stage four, greenhouseculturing and hardening. During stage one, initial tissue culturing, thetissue culture is established and certified contaminant-free. Duringstage two, the initial tissue culture is multiplied until a sufficientnumber of tissue samples are produced to meet production goals. Duringstage three, the tissue samples grown in stage two are divided and growninto individual plantlets. At stage four, the transformed plantlets aretransferred to a greenhouse for hardening where the plants' tolerance tolight is gradually increased so that it can be grown in the naturalenvironment.

Preferably, mature transformed plants generated as described above arefurther selected for abiotic stress tolerance. Accordingly, transformedand non-transformed (wild type) plants are exposed to an abiotic stresscondition, such as water depravation, suboptimal temperature, nutrientdeficiency, or preferably a salt stress condition. Salt stress can beeffected in many ways such as, for example, by irrigating the plantswith a hyperosmotic solution, by cultivating the plants hydroponicallyin a hyperosmotic growth solution (e.g., Hoagland solution), or byculturing the plants in a hyperosmotic growth medium (e.g., MS medium).Since different plants vary considerably in their tolerance to salinity,the salt concentration in the irrigation water, growth solution, orgrowth medium is preferably adjusted according to the specificcharacteristics of the specific plant cultivar or variety, so as toinflict a mild or moderate effect on the physiology and/or morphology ofthe plants (for guidelines as to appropriate concentration see,Bernstein and Kafkafi, Root Growth Under Salinity Stress In: PlantRoots, The Hidden Half 3rd ed. Waisel Y, Eshel A and Kafkafi U.(editors) Marcel Dekker Inc., New York, 2002, and reference therein).Following exposure to the stress condition the plants are frequentlymonitored until substantial physiological and/or morphological effectsappear in wild type plants. Subsequently, transformed plants notexhibiting substantial physiological and/or morphological effects, orexhibiting higher biomass than wild-type plants, are identified asabiotic stress tolerant plants.

Although stable transformation is presently preferred, transienttransformation of leaf cells, meristematic cells or the whole plant isalso envisaged by the present invention.

Transient transformation can be effected by any of the direct DNAtransfer methods described above or by viral infection using modifiedplant viruses.

Viruses that have been shown to be useful for the transformation ofplant hosts include CaMV, TMV and BV. Transformation of plants usingplant viruses is described in U.S. Pat. No. 4,855,237 (BGV), EP-A 67,553(TMV), Japanese Published Application No. 63-14693 (TMV), EPA 194,809(BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications inMolecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, NewYork, pp. 172-189 (1988). Pseudovirus particles for use in expressingforeign DNA in many hosts, including plants, is described in WO87/06261.

Preferably, the virus of the present invention is avirulent and thus isincapable of causing severe symptoms such as reduced growth rate,mosaic, ring spots, leaf roll, yellowing, streaking, pox formation,tumor formation and pitting. A suitable avirulent virus may be anaturally occurring avirulent virus or an artificially attenuated virus.Virus attenuation may be effected by using methods well known in the artincluding, but not limited to, sub-lethal heating, chemical treatment orby directed mutagenesis techniques such as described, for example, byKurihara and Watanabe (Molecular Plant Pathology 4:259-269, 2003),Gal-on et al. (1992), Atreya et al. (1992) and Huet et al. (1994).

Suitable virus strains can be obtained from available sources such as,for example, the American Type culture Collection (ATCC) or by isolationfrom infected plants. Isolation of viruses from infected plant tissuescan be effected by techniques well known in the art such as described,for example by Foster and Tatlor, Eds. “Plant Virology Protocols: FromVirus Isolation to Transgenic Resistance (Methods in Molecular Biology(Humana Pr), Vol 81)”, Humana Press, 1998. Briefly, tissues of aninfected plant believed to contain a high concentration of a suitablevirus, preferably young leaves and flower petals, are ground in a buffersolution (e.g., phosphate buffer solution) to produce a virus infectedsap which can be used in subsequent inoculations.

Construction of plant RNA viruses for the introduction and expression ofnon-viral exogenous polynucleotide sequences in plants is demonstratedby the above references as well as by Dawson, W. O. et al., Virology(1989) 172:285-292; Takamatsu et al. EMBO J. (1987) 6:307-311; French etal. Science (1986) 231:1294-1297; and Takamatsu et al. FEBS Letters(1990) 269:73-76.

When the virus is a DNA virus, suitable modifications can be made to thevirus itself. Alternatively, the virus can first be cloned into abacterial plasmid for ease of constructing the desired viral vector withthe foreign DNA. The virus can then be excised from the plasmid. If thevirus is a DNA virus, a bacterial origin of replication can be attachedto the viral DNA, which is then replicated by the bacteria.Transcription and translation of this DNA will produce the coat proteinwhich will encapsidate the viral DNA. If the virus is an RNA virus, thevirus is generally cloned as a cDNA and inserted into a plasmid. Theplasmid is then used to make all of the constructions. The RNA virus isthen produced by transcribing the viral sequence of the plasmid andtranslation of the viral genes to produce the coat protein(s) whichencapsidate the viral RNA.

Construction of plant RNA viruses for the introduction and expression inplants of non-viral exogenous polynucleotide sequences such as thoseincluded in the construct of the present invention is demonstrated bythe above references as well as in U.S. Pat. No. 5,316,931.

In one embodiment, a plant viral polynucleotide is provided in which thenative coat protein coding sequence has been deleted from a viralpolynucleotide, a non-native plant viral coat protein coding sequenceand a non-native promoter, preferably the subgenomic promoter of thenon-native coat protein coding sequence, capable of expression in theplant host, packaging of the recombinant plant viral polynucleotide, andensuring a systemic infection of the host by the recombinant plant viralpolynucleotide, has been inserted. Alternatively, the coat protein genemay be inactivated by insertion of the non-native polynucleotidesequence within it, such that a protein is produced. The recombinantplant viral polynucleotide may contain one or more additional non-nativesubgenomic promoters. Each non-native subgenomic promoter is capable oftranscribing or expressing adjacent genes or polynucleotide sequences inthe plant host and incapable of recombination with each other and withnative subgenomic promoters. Non-native (foreign) polynucleotidesequences may be inserted adjacent the native plant viral subgenomicpromoter or the native and a non-native plant viral subgenomic promotersif more than one polynucleotide sequence is included. The non-nativepolynucleotide sequences are transcribed or expressed in the host plantunder control of the subgenomic promoter to produce the desiredproducts.

In a second embodiment, a recombinant plant viral polynucleotide isprovided as in the first embodiment except that the native coat proteincoding sequence is placed adjacent one of the non-native coat proteinsubgenomic promoters instead of a non-native coat protein codingsequence.

In a third embodiment, a recombinant plant viral polynucleotide isprovided in which the native coat protein gene is adjacent itssubgenomic promoter and one or more non-native subgenomic promoters havebeen inserted into the viral polynucleotide. The inserted non-nativesubgenomic promoters are capable of transcribing or expressing adjacentgenes in a plant host and are incapable of recombination with each otherand with native subgenomic promoters. Non-native polynucleotidesequences may be inserted adjacent the non-native subgenomic plant viralpromoters such that the sequences are transcribed or expressed in thehost plant under control of the subgenomic promoters to produce thedesired product.

In a fourth embodiment, a recombinant plant viral polynucleotide isprovided as in the third embodiment except that the native coat proteincoding sequence is replaced by a non-native coat protein codingsequence.

The viral vectors are encapsidated by the coat proteins encoded by therecombinant plant viral polynucleotide to produce a recombinant plantvirus. The recombinant plant viral polynucleotide or recombinant plantvirus is used to infect appropriate host plants. The recombinant plantviral polynucleotide is capable of replication in the host, systemicspread in the host, and transcription or expression of foreign gene(s)(exogenous polynucleotide) in the host to produce the desired protein.

Techniques for inoculation of viruses to plants may be found in Fosterand Taylor, eds. “Plant Virology Protocols: From Virus Isolation toTransgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol81)”, Humana Press, 1998; Maramorosh and Koprowski, eds. “Methods inVirology” 7 vols, Academic Press, New York 1967-1984; Hill, S. A.“Methods in Plant Virology”, Blackwell, Oxford, 1984; Walkey, D. G. A.“Applied Plant Virology”, Wiley, New York, 1985; and Kado and Agrawa,eds. “Principles and Techniques in Plant Virology”, VanNostrand-Reinhold, New York.

In addition to the above, the polynucleotide of the present inventioncan also be introduced into a chloroplast genome thereby enablingchloroplast expression.

A technique for introducing exogenous polynucleotide sequences to thegenome of the chloroplasts is known. This technique involves thefollowing procedures. First, plant cells are chemically treated so as toreduce the number of chloroplasts per cell to about one. Then, theexogenous polynucleotide is introduced via particle bombardment into thecells with the aim of introducing at least one exogenous polynucleotidemolecule into the chloroplasts. The exogenous polynucleotides selectedsuch that it is integratable into the chloroplast's genome viahomologous recombination which is readily effected by enzymes inherentto the chloroplast. To this end, the exogenous polynucleotide includes,in addition to a gene of interest, at least one polynucleotide stretchwhich is derived from the chloroplast's genome. In addition, theexogenous polynucleotide includes a selectable marker, which serves bysequential selection procedures to ascertain that all or substantiallyall of the copies of the chloroplast genomes following such selectionwill include the exogenous polynucleotide. Further details relating tothis technique are found in U.S. Pat. Nos. 4,945,050; and 5,693,507which are incorporated herein by reference. A polypeptide can thus beproduced by the protein expression system of the chloroplast and becomeintegrated into the chloroplast's inner membrane.

Since abiotic stress tolerance in plants can involve multiple genesacting additively or in synergy (see, for example, in Quesda et al.,Plant Physiol. 130:951-063, 2002), the present invention also envisagesexpressing a plurality of exogenous polynucleotides in a single hostplant to thereby achieve superior abiotic stress tolerance.

Expressing a plurality of exogenous polynucleotides in a single hostplant can be effected by co-introducing multiple nucleic acidconstructs, each including a different exogenous polynucleotide, into asingle plant cell. The transformed cell can than be regenerated into amature plant using the methods described hereinabove.

Alternatively, expressing a plurality of exogenous polynucleotides in asingle host plant can be effected by co-introducing into a singleplant-cell a single nucleic-acid construct including a plurality ofdifferent exogenous polynucleotides. Such a construct can be designedwith a single promoter sequence which can transcribe a polycistronicmessage including all the different exogenous polynucleotide sequences.To enable co-translation of the different polypeptides encoded by thepolycistronic message, the polynucleotide sequences can be inter-linkedvia an internal ribosome entry site (IRES) sequence which facilitatestranslation of polynucleotide sequences positioned downstream of theIRES sequence. In this case, a transcribed polycistronic RNA moleculeencoding the different polypeptides described above will be translatedfrom both the capped 5′ end and the two internal IRES sequences of thepolycistronic RNA molecule to thereby produce in the cell all differentpolypeptides. Alternatively, the construct can include several promotersequences each linked to a different exogenous polynucleotide sequence.

The plant cell transformed with the construct including a plurality ofdifferent exogenous polynucleotides, can be regenerated into a matureplant, using the methods described hereinabove.

Alternatively, expressing a plurality of exogenous polynucleotides in asingle host plant can be effected by introducing different nucleic acidconstructs, including different exogenous polynucleotides, into aplurality of plants. The regenerated transformed plants can then becross-bred and resultant progeny selected for superior abiotic stresstolerance and/or biomass traits, using conventional plant breedingtechniques.

Hence, the present application provides methods of utilizing novelabiotic stress-tolerance genes to increase tolerance to abiotic stressand/or biomass and/or yield in a wide range of economical plants, safelyand cost effectively.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-IIIColigan J. E., ed. (1994); Stites et al. (eds), “Basic and ClinicalImmunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994);Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W.H. Freeman and Co., New York (1980); available immunoassays areextensively described in the patent and scientific literature, see, forexample, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578;3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533;3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;“Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic AcidHybridization” Hames, B. D., and Higgins S. J., eds. (1985);“Transcription and Translation” Hames, B. D., and Higgins S. J., eds.(1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “ImmobilizedCells and Enzymes” IRL Press, (1986); “A Practical Guide to MolecularCloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317,Academic Press; “PCR Protocols: A Guide To Methods And Applications”,Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategiesfor Protein Purification and Characterization—A Laboratory CourseManual” CSHL Press (1996); all of which are incorporated by reference asif fully set forth herein. Other general references are providedthroughout this document. The procedures therein are believed to be wellknown in the art and are provided for the convenience of the reader.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below.

Example 1 Identifying Putative Abiotic Stress Tolerance Genes

Putative abiotic stress-tolerance (ABST) genes were selected from NCBIdatabases of tomato expressed sequence tags (ESTs) and cDNAs. Thedatabase sequences were clustered and assembled using the LEADS™software (Compugen). Clustering resulted in more than 20,000 clusters,each representing a different gene. An expression profile summary wascompiled for each cluster by pooling all keywords included in thesequence records comprising the cluster. The clusters were then screenedto include polynucleotides originating from libraries identified bykeywords relating to ABST. The selected clusters were further filteredto exclude any cluster which included more than 100 ESTs per clusterand/or any cluster in which less than 50% of the sequences wereannotated by ABST-related keywords.

Prior art ABST plant genes were identified from the publications ofQuesada et al. (Plant Physiol. 130:951-963, 2002); Apse and Blumwald(Curr Opin Biotechnol. 13:146-150, 2002); Rontein et al. (Metab Eng4:49-56, 2002); and references therein. Known plant ABST genes werealigned with the clustered tomato nucleic-acid sequences using the BLASTprogram. The tomato sequences having an e-score value lower than 5 wereidentified as ABST orthologes. Additional prior art tomato ABST geneswere identified by searching the clustered tomato sequence records usingthe keywords “root”, “crown gall”, “nutrient”, “callus”, “disease”,“pathogen”, “elicitor” and “pseudomonas”.

Finally, all identified prior art ABST genes were matched (by sequencealignment using the BLAST software) with the output set of tomato geneclusters, selected as described above. Consequently, about 40% of thegenes selected in the output set of clusters which matched with priorart ABST genes proved to be known ABST genes, indicating that theremaining genes of the selected clusters are potentially capable ofincreasing abiotic stress tolerance in plants.

The selected polynucleotide sequences (Table 1A), were analyzed forpresence of ORFs using Vector NTI suite (InforMax, U.K.) version 6(Hasting Software, Inc: World Wide Web (dot) Generunner (dot) com/).ORFs identified in each of these polynucleotide sequences were comparedto Genbank database sequences, using Blast (World Wide Web (dot) ncbi(dot) nlm (dot) nih (dot) gov/BLAST/); the ORF displaying the highesthomology to a GenBank sequence or sequences, was mapped in order toidentify an ATG start codon. The position of the ATG start codon of thisORF was then compared with that of the identified polynucleotidesequence in order to verify that each of the eighteen sequencesdescribed herein includes a full length ORF and an ATG start codon (thusqualifying as a “putative ABST gene”).

TABLE 1A Putative ABST genes ABST No. SEQ ID No.  1 1  3 2  5 3  6 4 105 11 6 12 7 19 8 22 9 24 10 26 11 27 12 36 13 37 14 39_T0 15 39_T1 1649_T0 17 49_T1 18

ABST polypeptide homologues were identified from the NCBI databasesusing BLAST software (Table 1B).

TABLE 1B ABST homologues ABST ABST Polypeptide ABST Polypeptide ProteinPutative Gene Homologue Homologue Homology SEQ ID No. NCBI Accession No.SEQ ID No. (%) 1 BAA96366 39 98 1 AAS47510 40 98 1 NP_567151 41 97 1NP_567104 42 96 1 AAK55664 43 96 1 P46298 44 97 1 T01338 45 96 1 T4788846 95 1 BAD09465 47 92 1 Q05761 48 91 1 BAD09464 49 88 1 CAA79496 50 841 EAJ94592 51 85 4 NP_188036 52 76 4 NP_035977 53 70 4 XP_342608 54 69 4T09295 55 60 4 NP_564717 56 59 4 AAM63624 57 59 9 P37707 58 93 9CAD37200 59 81 9 CAA04664 60 78 9 AAM64572 61 77 9 NP_189345 62 77 9NP_974979 63 60 13 AAC49992 64 88 13 T10804 65 87 13 AAL38357 66 87 13NP_188245 67 87 13 NP_193465 68 87 13 AAG44945 69 86 13 T07819 70 86 13T12632 71 86 13 CAC39073 72 86 13 T01648 73 86 13 AAF90121 74 86 13S48116 75 86 13 AAO86710 76 86 13 T14002 77 85 13 T14001 78 85 13 T4888679 85 13 T14314 80 85 13 P33560 81 85 13 P21653 82 85 13 T14000 83 85 13T48884 84 85 13 P24422 85 85 13 AAB53329 86 85 14 NP_200279 87 67 14AAM64276 88 67 14 AAO72577 89 66 14 NP_175518 90 64 14 BAC78588 91 64 14BAD03011 92 62

Five of these genes were selected as having the most potential of beingputative ABS tolerance genes on the basis of digital expression profiles(i.e. known to be up-regulated under different stress conditions) aslisted in Tables 2-6, and homologies to public protein sequences anddomains through multi sequence alignment searches. Also, only genes withlow and medium expression levels were included.

The five genes are listed together with their functions in Table 1Cbelow.

TABLE 1C Selected Putative ABST genes Gene Length of Protein Proteindomain name AC number, CDS bps length aa Similarities Go classificationABST_1 BG123819/ 833 151 Ribosomal RNA binding, SEQ ID TC153989 proteinS13 morphogenesis, protein NO: 1 biosynthesis in cytosol ABST_6BG627487/ 1242 163 DnaJ domain Chaperone activity, SEQ ID TC162949protein folding NO: 4 ABST_22 BG127611/ 1518 311 Asparagine-rich UnknownSEQ ID TC154372 region profile NO: 9 ABST_36 AA824892/ 1466 249 Majorintrinsic Water channel, SEQ ID TC154006 protein endomembrane activityNO: 13 ABST_37 AW220029/ 1245 244 Helix-loop-helix DNA binding, SEQ IDTC156100 DNA-binding transcription NO: 14 domain factor activity

Digital expression, also known as electronic northern blot, is a toolfor virtually displaying the expression profile of query genes based onthe EST sequences forming the cluster. The tool can provide theexpression profile of a cluster in terms of plant anatomy (in whattissues/organs is the gene expressed), developmental stage (thedevelopmental stages at which a gene can be found) and profile oftreatment (provides the physiological conditions under which a gene isexpressed such as drought, cold, pathogen infection, etc). Given arandom distribution of ESTs in the different clusters, the digitalexpression provides a probability value that describes the probabilityof a cluster having a total of N ESTs to contain X ESTs from a certaincollection of libraries. For the probability calculations, variousconsiderations are taken: a) the number of ESTs in the cluster, b) thenumber of ESTs of the implicated and related libraries, c) the overallnumber of ESTs available representing the species. Thereby clusters withlow probability values are highly enriched with ESTs from the group oflibraries of interest indicating a specialized expression.

The digital expression profile for ABST_1 (SEQ ID NO:1) is summarizedbelow in Table 2.

TABLE 2 Change in expression of ABST_1 due to exposure to varioustreatments ESTs in ESTs in Expected Keyword Gene Production ESTs Foldp-value hormone 3 23195 7.14655 0.419783 0.978926 treatment Elicitorsand 1 14540 4.47988 0.22322 0.990341 pathogens mix Mix of 2 8655 2.666670.749999 0.751616 elicitors nutrient 1 3258 1.00381 0.9962 0.636393deficiencies —N, —P, —K, 1 3258 1.00381 0.9962 0.636393 —Fe, —Alpathogen 13 30639 9.4401 1.3771 0.141666 Agrobacterium 4 5107 1.57352.5421 0.0728454 tumefaciens C58 CONTROL 1 419 1 1 0.12124 Ralstonia s.Elicitors and 1 14540 4.47988 0.22322 0.990341 pathogens mix

The digital expression profile for ABST_6 (SEQ ID NO:4) is summarizedbelow in Table 3.

TABLE 3 Change in expression of ABST_6 due to exposure to varioustreatments ESTs in ESTs in Expected Keyword Gene Production ESTs Foldp-value hormone 3 23195 2.59875 1.1544 0.489866 treatment Mix of 3 86551 3 0.0509528 elicitors pathogen 2 30639 3.43276 0.582621 0.876812pseudomonas 2 10177 1.14022 1.75405 0.316866 syringae

The digital expression profile for ABST_22 (SEQ ID NO:9) is summarizedbelow in Table 4.

TABLE 4 Change in expression of ABST_22 due to exposure to varioustreatments ESTs in ESTs in Expected Keyword Gene Production ESTs Foldp-value hormone 2 23195 5.7389 0.348499 0.982893 treatment Mix of 2 86552.14142 0.933961 0.636873 elicitors nutrient 3 3258 1 3 0.0469483deficiencies —N, —P, —K, 3 3258 1 3 0.0469483 —Fe, —Al pathogen 6 306397.58069 0.791485 0.788347 Agrobacterium 2 5107 1.26357 1.58282 0.361357tumefaciens C58

The digital expression profile for ABST_36 (SEQ ID NO:13) is summarizedbelow in Table 5.

TABLE 5 Change in expression of ABST_36 due to exposure to varioustreatments ESTs ESTs in in Pro- Expected Keyword Gene duction ESTs Foldp-value heavy metals 1 741 1 1 0.307469 0.2 mM CdCl2 1 476 1 1 0.210111hormone 12 23195 11.4778 1.0455 0.480794 treatment Elicitors and 4 145407.19496 0.555945 0.934556 pathogens mix Mix of 8 8655 4.28283 1.867920.0656842 elicitors nutrient 9 3258 1.61219 5.58248 3.76416E−05deficiencies —N, —P, —K, 9 3258 1.61219 5.58248 3.76416E−05 —Fe, —Alpathogen 17 30639 15.1614 1.12127 0.344706 Agrobacterium 3 5107 2.527141.18711 0.464783 tumefaciens C58 Elicitors and 4 14540 7.19496 0.5559450.934556 pathogens mix pseudomonas 10 10177 5.03598 1.98571 0.0295878syringae

The digital expression profile for ABST_37 (SEQ ID NO:14) is summarizedbelow in Table 6.

TABLE 6 Change in expression of ABST_37 due to exposure to varioustreatments ESTs in ESTs in Expected Keyword Gene Production ESTs Foldp-value hormone 1 23195 1 1 0.643514 treatment Mix of 1 8655 1 1 0.31009elicitors nutrient 3 3258 1 3 0.000275679 deficiencies —N, —P, —K, 33258 1 3 0.000275679 —Fe, —Al

Example 2 In-Situ Validation/Expression Studies

The five genes listed in Table 1C were validated in situ as putativeABST genes by analyzing their expression profile in tomato plants underfavorable, salt and drought stress conditions.

The expression studies were carried out on three tomato lines—sensitivetomato variety (Evoline 1), processing tomato line with moderatetolerance to salt (Evoline 3, also referred to herein as M82) anddrought and high salt tolerant tomato line (Evoline 2). All lines weretested for several seasons for their levels of tolerance to salt andother soil stresses such as drought. All lines are commerciallyavailable from Evogene, Rehovot, Israel.

Methods

Salt Induction:

Salt stress induction was performed by introducing the roots of 14 daysold tomato seedlings, of different tomato homozygote varieties, into awater bath which contained a solution of Hogland (comprising KNO₃—8 mM,MgSO₄—1 mM, KH₂PO₄—1 mM, and microelements, all dissolved in water, atpH 6.5), and 300 mM NaCl. Plants were placed on a floating tray, suchthat only the roots were dipped in the solution. Plants were grown insalt solution for 5 weeks during which the degree of tolerance to thesalt stress was measured, by comparing plant development and biomass.The experiment was performed in 3 sequential seasons and with 5 repeatsfor each line in each experiment. The experiments identified 3 tomatolines showing consistent level of either weak (Evoline1), moderate(Evoline3), or high (Evoline2) level of tolerance. During the lastexperiment RNA samples from leaves and roots from Evoline1 and Evoline3were taken 5, 9, 72, 240 hours after introducing the plants to saltsolution.

Drought Induction:

Levels of tolerance to drought induction were tested on Evoline 1,Evoline2, and Evoline3 tomato plants. The plants were grown in CYGGermination Pouches, (Mega International, MN, USA), from germinationuntil 4 true leaf stage in a regular nutrient solution. Droughtconditions were applied by adding polyethylene glycol-PEG to the growthsolution to a final concentration of 15%. RNA samples from leaves androots were taken at time 0, 0.5 h, 3 h, 6 h and 48 h following droughtinduction. RNA expression level was measured by using quantitative RTPCR.

Results

As illustrated in FIG. 2, seedlings of the sensitive line (Evoline 1)were much smaller with far fewer leaves than seedlings of the tolerantline (Evoline 2) following four weeks growth under water irrigationcontaining 300 mM NaCl.

Tables 7-9 below summarise the up-regulation change in gene expressionin response to various stress conditions (e.g. drought and salt) ofpolynucleotides of the present invention in tomato plants as calculatedby quantitative RT-PCR.

TABLE 7 Relative expression of the ABST genes following salt inductioncompared to their expression at time 0 (T0) in leaves and roots of atomato tolerant line (Evoline 2 = highly tolerant line) Time afterinduction (hours) Organ ABST_1 ABST_6 ABST_19 ABST_22 ABST_27 ABST_36ABST_37 0 leaves 1.00 1.00 1.00 1.00 1.00 1.00 1.00 5 leaves 1.32 0.561.19 1.33 1.61 1.97 1.63 9 leaves 1.54 0.48 0.99 1.51 1.50 1.17 1.76 72leaves 1.26 0.30 1.13 1.75 1.22 0.57 3.60 240 leaves 0.54 1.89 1.22 0.620.46 0.74 8.05 0 roots 1.00 1.00 1.00 1.00 1.00 1.00 1.00 5 roots 0.891.23 0.89 1.09 0.66 0.57 1.20 9 roots 0.72 1.03 1.01 0.78 0.69 0.42 0.8372 roots 0.81 2.06 1.24 1.38 0.97 0.29 1.15 240 roots 0.50 7.91 1.700.66 0.57 0.10 4.77

TABLE 8 Relative expression of the ABST genes following salt inductioncompared to their expression in leaves and roots of tomato sensitivevariety (Evoline 1 = salt sensitive line) Time after induction (hours)Organ ABST_1 ABST_6 ABST_19 ABST_22 ABST_27 ABST_36 ABST_37 0 leaves1.00 1.00 1.00 1.00 1.00 1.00 1.00 5 leaves 1.60 0.00 1.10 1.18 1.581.18 0.00 9 leaves 0.88 0.34 1.14 0.83 1.80 0.58 0.83 72 leaves 0.700.20 1.21 0.81 0.95 0.40 1.71 240 leaves 0.44 2.25 1.56 0.69 0.56 0.237.63 0 roots 1.00 1.00 1.00 1.00 1.00 1.00 1.00 5 roots 0.96 1.56 1.051.01 1.25 0.84 0.81 9 roots 0.84 1.10 1.08 0.58 1.09 0.46 0.47 72 roots0.83 1.31 1.43 1.00 1.41 0.24 0.44 240 roots 0.59 9.13 2.03 0.77 1.000.07 1.77

TABLE 9 Relative expression of the ABST genes following droughtinduction compared to their expression in T0 in leaves and roots ofseedlings of processing tomato variety (Evoline 3 = moderate salttolerant line-M82) Time after induction (h) Organ ABST_1 ABST_6 ABST_19ABST_22 ABST_27 ABST_36 ABST_37 0 Roots 1.00 1.00 1.00 1.00 1.00 1.001.00 0.5 Roots 0.62 0.24 0.94 0.58 0.81 0.40 0.51 3 Roots 0.40 0.21 0.770.40 0.54 0.25 0.26 6 Roots 0.58 0.13 1.15 0.36 0.82 0.24 0.29 48 Roots0.72 0.15 1.19 1.43 3.05 0.49 1.30 0 Leaves 1.00 1.00 1.00 1.00 1.001.00 1.00 0.5 Leaves 2.21 2.84 0.86 2.49 1.37 1.18 0.75 3 Leaves 2.243.83 1.40 2.97 0.84 2.55 0.81 6 Leaves 3.29 4.99 1.68 6.47 6.18 2.081.91 48 Leaves 2.70 4.99 1.20 8.80 3.60 0.46 1.88

As seen from tables 7-9, ABST_1, 6, 22, 27, 36 and 37 all showedinduction of expression under different stress conditions. The changesin gene expression as a response to various stresses could be classifiedinto two main categories—immediate up-regulation e.g. ABST_36 and ABST_1(from 30 minutes to 6 hours following induction) and delayedup-regulation e.g. ABST_37 and ABST_6 (24 to 240 hours followinginduction).

The expression profile of most the genes was also affected by thegenotype of the plants (highly tolerant line vs. sensitive line) and thespecific stress the plants were exposed to (i.e. salt or PEG). Threegenes showed changes in expression in leaves only (ABST_1, 22, 36). OnlyABST_6 and 37 showed up-regulation of expression in both leaves androots.

Significant changes were not detected in the expression of the ABST_19as a response to salt or drought stress.

Table 10 below summarises the main expression modes of the ABST genes ofthe present invention under salt and osmotic stress.

TABLE 10 Expression modes of the ABST genes of the present inventionunder salt and osmotic stresses Salt stress Osmotic Salt stress Osmoticstress (100 mM NaCl) stress (100 mM NaCl) (10% PEG) Gene name/ Evoline 1Evoline 2 Evoline 3 Evoline 1 Evoline 2 Evoline 3 time range Leaf RootABST_1 Up × 1.5 Up × 1.5 Up × 3 Stable Stable Down × 2 Early responseABST_1 Down × 2 Down × 2 Stable Down × 2 Down × 2 Stable Early responseABST_6 Down × 3 Down × 2 Up × 5 Stable Stable Down × 7 Early responseABST_6 Up × 2 Up × 2 Up × 5 Up × 9 Up × 8 Down × 7 Late response ABST_22Stable Up × 1.5 Up × 6 Stable Stable Down × 3 Early response ABST_22Stable Stable Up × 8 Stable Stable Stable Late response ABST_36 StableUp × 2 Up × 2 Down × 2 Down × 2 Down × 5 Early response ABST_36 Down × 5Stable Down × 2 Down × 5 Down × 3 Down × 2 Late response ABST_37 StableUp × 2 Stable Down × 2 Stable Down × 3 Early response ABST_37 Up × 8 Up× 8 Up × 2 Up × 1.5 Up × 5 Stable Late response Evoline 1: has low salttolerance Evoline 2: has high salt tolerance Evoline 3: has moderatesalt tolerance Early response: 0.5 hours to 9 hours from induction Lateresponse: 48 hours to 240 hours from induction

Example 3 Isolation of ABS Tolerance Genes of the Present Invention

RNA was extracted from 4 week-old tomato root and leaf tissues using TriReagent (Molecular Research Center, Inc), following the protocolprovided by the manufacturer (World Wide Web (dot) Mrcgene (dot) com/tri(dot) htm). Complementary DNA molecules were produced from the extractedmRNA using M-MuLV reverse-transcriptase (RT) enzyme (Roche) and T16NNDNA primer, according to the manufacturer's instructions. The cDNAsequences set forth in SEQ ID NOs: 1, 4, 8-9 and 12-14, were amplifiedby PCR using the primers described in Table 11 below, with PFU proofreading DNA polymerase enzyme (Promega-World Wide Web (dot) Promega(dot) com/pnotes/68/7381_07/7381_07 (dot) html), following the protocolprovided by the manufacturer. Additional restriction endonuclease siteswere added to the 5′ prime end of each primer to facilitate cloning ofthe ABS tolerance genes of the present invention in binary vectors.

TABLE 11 PCR primers used for amplifying ABS tolerance (ABST) genes ofthe present invention upstream downstream ABST gene Forward PrimerReverse Primer restriction restriction SEQ ID No SEQ ID No SEQ ID Nosite site 1 21 22 BamH1 SacI 4 23 24 BamH1 SacI 8 25 26 BamH1 SacI 9 2728 XbaI SmaI 12 29 30 BamH1 SacI 13 31 32 BamH1 SacI 14 33 34 BamH1 SmaI

Example 4 Cloning the ABST Genes of the Present Invention

The resulting PCR blunt ended products were purified using PCRPurification Kit (Qiagen, Germany), digested with the appropriaterestriction enzymes (Roche) and then inserted into the binary plasmidvector pPI. The plasmid pPI was constructed by inserting a syntheticpoly-(A) signal sequence, originating from pGL3 basic plasmid vector(Promega, Acc No U47295; by 4658-4811) into the HindIII restriction siteof the binary vector pBI101.3 (Clontech, Acc. No. U12640).

The resulting pPI plasmid was digested with restriction enzymes (BamHIand SacI; MBI Fermentas) and purified using PCR Purification Kit(Qiagen, Germany). The open pPI construct was then ligated with each ofthe seven PCR products described hereinabove. The ligation was effectedusing a ligation mixture containing T4 DNA ligase enzyme (Roche) and wasperformed according to the manufacturer's instructions.

The pPI constructs harboring ABST genes of the present invention wereintroduced to E. coli DH5 competent cells by electroporation, using aMicroPulser electroporator (Biorad), 0.2 cm cuvettes (Biorad) and EC-2electroporation program (Biorad). The treated cells were cultured in LBliquid medium at 37° C. for 1 hr, then plated over LB agar supplementedwith kanamycin (50 mg/L; Sigma) and incubated at 37° C. for 16 hrs.Colonies which developed on the selective medium were analyzed by PCRusing the primers set forth in SEQ ID NOs: 35-36, which were designed tospan the inserted sequence in the pPI plasmid. The resulting PCRproducts were separated on 1.5% agarose gels and the DNA fragment havingthe predicted size were isolated and sequenced using the ABI 377sequencer (Amersham Biosciences Inc) in order to verify that the correctDNA sequences were properly introduced to the E. coli cells.

Example 5 Generating Binary Vectors Comprising ABST Genes of the PresentInvention and Plant Promoters Operably Linked Thereto

Generating Binary Vectors Comprising the Cauliflower Mosaic Virus 35SPromoter:

The Cauliflower Mosaic Virus 35S promoter sequence (set forth in SEQ IDNO: 19) was inserted upstream of the ABST genes of the present inventionin each of the pPI constructs described above. The promoter was isolatedfrom the pBI121 plasmid (Clontech, Accession No. AF485783) using therestriction endonucleases HindIII and BamHI (Roche). The isolatedpromoter was ligated into the pPI constructs digested with the sameenzymes. Altogether, seven pPI constructs were generated, eachcomprising the CaMV 35S promoter positioned upstream of the ABST genesof the present invention having a sequence set forth in SEQ ID NO: 1, 4,8, 9, 12, 13 or 14.

Generating Binary Vectors Comprising the At6669 Promoter:

The At6669 promoter sequence (set forth in SEQ ID NO: 20) was insertedupstream of the ABST genes of the present invention in each of the pPIbinary constructs described above. The promoter was isolated fromArabidopsis thaliana var Col0 genomic DNA by PCR amplification using theprimers set forth in SEQ ID NOs: 37-38. The PCR product was purified(Qiagen, Germany) and digested with the restriction endonucleasesHindIII and BamHI (Roche). The resulting promoter sequence wasintroduced into the open binary constructs digested with the sameenzymes. Altogether, seven pPI constructs were generated, eachcomprising the At6669 promoter positioned upstream of the ABST genes ofthe present invention having a sequence set forth in SEQ ID NO: 1, 4, 8,9, 12, 13 or 14.

Example 6 Confirming At6669 Promoter Activity in Transgenic Arabidopsisthaliana

The capacity of At-6669 promoter to regulate transcription of genescarried by the pPI vector in plants was tested. Accordingly, thepromoter At6669 was inserted into the pPI binary vector upstream of aLuciferase reporter gene. The binary vector was introduced toArabidopsis thaliana plants using the procedure as described in Example6 below. Mature transformed T₂ Arabidopsis plants were assayed forbio-illumination in a darkroom using an ultra-low light detection camera(Princeton Instruments Inc., USA) using the procedure described byMeissner et al. (Plant J. 22:265, 2000). Illumination indicatingpositive Luciferase activity was observed in the flower and rootmeristem tissues of transformed plants (FIGS. 3A-3D).

To study the regulation mode of the promoter under stress conditions,the 6669 promoter and 35S promoter were both fused to the ArabidopsisRab7 gene. Rab7 expression under the 6669 promoter gave significantlyhigher salt and osmotic tolerance performance compared to 35S inArabidopsis, as determined by vegetative growth.

The promoter was further validated by comparing tolerance level ofArabidopsis plants containing the ABST genes of the present inventionunder the regulation of 6669 and under the regulation of 35S promoter(FIG. 4). The plants were transformed with seven ABST genes of thepresent invention (ABST_1, 6, 19, 22, 27, 36, 37) as described inExample 8. As illustrated in FIG. 4, the plant dry weight increasedfollowing transformation of the ABST genes under the 6669 promoter to agreater extent than following transformation of the ABST genes under the35S promoter both under normal and stress (100 mM salt) conditions.

Example 7 Transforming Agrobacterium tumefaciens Cells with BinaryVectors Harboring ABST Genes of the Present Invention

Each of the binary vectors described in Example 5 above were used totransform Agrobacterium cells. Two additional binary constructs, havingthe Luciferase reporter gene replacing an ABST gene (positioneddownstream of the 35S or At6669 promoter), were used as negativecontrols.

The binary vectors were introduced to Agrobacterium tumefaciens GV301,or LB4404 competent cells (about 10⁹ cells/mL) by electroporation. Theelectroporation was effected by using a MicroPulser electroporator(Biorad), 0.2 cm cuvettes (Biorad) and EC-2 electroporation program(Biorad). The treated cells were cultured in LB liquid medium at 28° C.for 3 hr, then plated over LB agar supplemented with gentamycin (50mg/L; for Agrobacterium strains GV301) or streptomycin (300 mg/L; forAgrobacterium strain LB4404) and kanamycin (50 mg/L) at 28° C. for 48hrs. Agrobacterium colonies which developed on the selective media wereanalyzed by PCR using the primers set forth in SEQ ID NOs: 35-36, whichwere designed to span the inserted sequence in the pPI plasmid. Theresulting PCR products were isolated and sequenced as described inExample 5 above, to verify that the correct ABST sequences were properlyintroduced to the Agrobacterium cells.

Example 8 Transformation of Arabidopsis thaliana Plants with ABST Genesof the Present Invention

Arabidopsis thaliana Columbia plants (T₀ plants) were transformed usingthe Floral Dip procedure described by Clough and Bent (10) and byDesfeux et al. (11), with minor modifications. Briefly, T₀ Plants weresown in 250 ml pots filled with wet peat-based growth mix. The pots werecovered with aluminum foil and a plastic dome, kept at 4° C. for 3-4days, then uncovered and incubated in a growth chamber at 18-24° C.under 16/8 hr light/dark cycles. The T₀ plants were ready fortransformation six days before anthesis.

Single colonies of Agrobacterium carrying the binary constructs,generated as described in Example 6 above, were cultured in LB mediumsupplemented with kanamycin (50 mg/L) and gentamycin (50 mg/L). Thecultures were incubated at 28° C. for 48 hrs under vigorous shaking andthen centrifuged at 4000 rpm for 5 minutes. The pellets comprisingAgrobacterium cells were re-suspended in a transformation mediumcontaining half-strength (2.15 g/L) Murashig-Skoog (Duchefa); 0.044 μMbenzylamino purine (Sigma); 112 μg/L B5 Gambourg vitamins (Sigma); 5%sucrose; and 0.2 ml/L Silwet L-77 (OSI Specialists, CT) indouble-distilled water, at a pH of 5.7.

Transformation of T₀ plants was effected by inverting each plant into anAgrobacterium suspension, such that the above ground plant tissue wassubmerged for 3-5 seconds. Each inoculated T₀ plant was immediatelyplaced in a plastic tray, then covered with a clear plastic dome tomaintain humidity and was kept in the dark at room temperature for 18hrs, to facilitate infection and transformation. Transformed(transgenic) plants were then uncovered and transferred to a greenhousefor recovery and maturation. The transgenic T₀ plants were grown in thegreenhouse for 3-5 weeks until siliques were brown and dry. Seeds wereharvested from plants and kept at room temperature until sowing.

For generating T₁ and T₂ transgenic plants harboring the genes, seedscollected from transgenic T₀ plants were surface-sterilized by soakingin 70% ethanol for 1 minute, followed by soaking in 5% sodiumhypochloride and 0.05% triton for 5 minutes. The surface-sterilizedseeds were thoroughly washed in sterile distilled water then placed onculture plates containing half-strength Murashig-Skoog (Duchefa); 2%sucrose; 0.8% plant agar; 50 mM kanamycin; and 200 mM carbenicylin(Duchefa). The culture plates were incubated at 4° C. for 48 hours thentransferred to a growth room at 25° C. for an additional week ofincubation. Vital T₁ Arabidopsis plants were transferred to a freshculture plates for another week of incubation. Following incubation, theT₁ plants were removed from culture plates and planted in growth mixcontained in 250 ml pots. The transgenic plants were allowed to grow ina greenhouse to maturity. Seeds harvested from T₁ plants were culturedand grown to maturity as T₂ plants under the same conditions as used forculturing and growing the T₁ plants.

Example 9 Evaluating Growth of Transgenic Plants Cultivated UnderAbiotic Stress Conditions

Methods:

T₁ or T₂ transgenic plants generated as described above wereindividually transplanted into pots containing a growth mixture of peatand vermiculite (volume ratio 3:2, respectively). The pots were coveredfor a 24 hr period for hardening, then placed in the greenhouse inrandom order and irrigated with tap water (provided from the pots'bottom every 3-5 days) for seven days. Thereafter, half of the plantswere irrigated with a salt solution (100 mM NaCl and 5 mM CaCl₂) toinduce salinity stress (stress conditions). The other half was irrigatedwith tap water throughout (normal conditions). All plants were grown inthe greenhouse at 100% RH for 28 days and then harvested (the aboveground tissue).

Vigor Measurement:

Fresh and dry mass were measured as a function of plant vigor. Dry masswas measured immediately following drying in an oven at 50° C. for sevendays.

Results:

Fresh Weight:

No significant differences in plant fresh weights were observed betweenthe T₁ plants transformed with 3 different ABST genes and plantstransformed with the Luciferase reporter gene, grown either under normalor stress conditions (FIG. 5 and Table 12 below). Yet, T₁ plantstransformed with SEQ ID NO: 1 positioned under the regulatory control ofthe At6669 promoter maintained 71% of their fresh weight when exposed tostress conditions, while the control plants (carrying Luciferase genepositioned under the regulatory control of the AT6669 promoter)maintained only 61% of their fresh weight under similar stressconditions.

TABLE 12 Fresh weight of T₁ transgenic Arabidopsis plants irrigated withwater or salt solution Transgene Irrigation (SEQ solution ID NO)Promoter N Rows¹ (mM NaCl) Mean (g) Std Error Luciferase At6669 2 00.7925 0.0275 Luciferase At6669 2 100 0.485 0.045 13 At6669 8 0 0.816250.020305 13 At6669 8 100 0.4725 0.029246 1 At6669 8 0 0.7875 0.026032 1At6669 8 100 0.55875 0.044699 8 At6669 8 0 0.8575 0.023088 8 At6669 8100 0.440625 0.011198 ¹N Rows represent number of independenttransformation event plants measured. For each transgene, 3-5independent transformation events with 1-3 plants per a singletransformation event were used.

T₂ plants transformed with SEQ ID NOs: 7 or 14 positioned under theregulatory control of the 35S promoter accumulated significantly higherbiomass than control plants, regardless of growth conditions. As shownin FIG. 6A and Table 13 below, the mean fresh weight of plantstransformed with SEQ ID NOs: 7 and 14, grown under stress conditions,were 15% and 24%, respectively, higher than the mean fresh weightcontrol plants grown under similar stress conditions. Similarly, themean fresh weight of plants transformed with SEQ ID NOs: 7 or 14, grownunder normal conditions, were 21% and 27%, respectively, higher than themean fresh weight control plants grown under similar normal conditions.

Similar phenomenon was observed with T₂ plants transformed with SEQ IDNO: 4 positioned under the regulatory control of the 35S promoter.Accordingly, as shown in FIG. 6A and Table 13 below, the mean freshweight of plants transformed with SEQ ID NO: 4 was 14% and 7% was higherthan the mean fresh weight of control plants grown under stress andnormal conditions, respectively. Similarly, T₂ plants transformed withSEQ ID NO: 4 positioned under the regulatory control of the At6669promoter exhibited 1.3 and 5% higher biomass than control plants grownunder stress and normal conditions, respectively (Table 14). However,these differences were not found statistically different under theexperimental conditions.

TABLE 13 Fresh weight of T₂ transgenic Arabidopsis plants irrigated withwater or salt solution Transgene Irrigation (SEQ solution ID NO)Promoter N Rows (mM NaCl) Mean (g) Std Error Luciferase CaMV-35S 11 00.352727 0.011208 Luciferase CaMV-35S 11 100 0.280909 0.010484 9CaMV-35S 11 0 0.426364 0.019599 9 CaMV-35S 11 100 0.322727 0.027306 12CaMV-35S 11 0 0.374545 0.015746 12 CaMV-35S 11 100 0.249091 0.020647 1CaMV-35S 8 0 0.36625 0.034171 1 CaMV-35S 8 100 0.265 0.031225 13CaMV-35S 11 0 0.349091 0.013515 13 CaMV-35S 11 100 0.293636 0.019921 14CaMV-35S 11 0 0.446364 0.025558 14 CaMV-35S 11 100 0.348182 0.023772 8CaMV-35S 11 0 0.310909 0.015223 8 CaMV-35S 11 100 0.253636 0.01539 4CaMV-35S 11 0 0.379091 0.010992 4 CaMV-35S 11 100 0.318182 0.013336 ¹NRows represent number of independent transformation event plantsmeasured. For each transgene, 3-5 independent transformation events with1-3 plants per a single transformation event were used.

T₂ plants transformed with SEQ ID NOs: 1 and 13 positioned under theregulatory control of the At6669 promoter and grown under stressconditions, exhibited significantly higher biomass than control plantsgrown under similar stress conditions. The mean fresh weight of T₂plants transformed with SEQ ID NOs: 1 and 13 positioned under theregulatory control of the At6669 promoter, and grown under stressconditions, were 37% and 21%, respectively, higher than the mean freshweight control plants grown under similar stress conditions (FIG. 6B andTable 14 below). No significant increase in biomass over control wasobserved when these transgenic plants (carrying SEQ ID NOs: 1 and 13regulated under At6669 promoter) where grown under normal conditions.

TABLE 14 Fresh weight of T₂ transgenic Arabidopsis plants irrigated withwater or salt solution Transgene Irrigation (SEQ solution (mM ID NO)Promoter N Rows NaCl) Mean (g) Std Error Luciferase At6669 6 0 0.30.010328 Luciferase At6669 6 100 0.125 0.009916 13 At6669 6 0 0.2866670.024449 13 At6669 6 100 0.151667 0.007032 1 At6669 6 0 0.305 0.03423 1At6669 6 100 0.171667 0.012225 4 At6669 6 0 0.315 0.049983 4 At6669 6100 0.126667 0.005578 12 At6669 6 0 0.263333 0.012824 12 At6669 6 1000.098333 0.007923 8 At6669 6 0 0.228333 0.020235 8 At6669 6 100 0.1216670.004014 ¹N Rows represent number of independent transformation eventplants measured. For each transgene, 3-5 independent transformationevents with 1-3 plants per a single transformation event were used.

The results illustrate that the isolated ABST genes of the presentinvention, set forth in SEQ ID NOs: 1 and 13, are capable of increasingplant tolerance to abiotic stress, such as a salinity stress. Inaddition, the isolated ABST genes of the present invention as set forthin SEQ ID NOs: 7, 14 (and possibly also 4), are capable of substantiallypromoting biomass in plants grown under stress, as well as under normalconditions.

Dry Mass:

Table 15 summarises the results the dry mass of T1 plants grown under100 mM NaCl.

TABLE 15 Dry mass of T1 plants grown under 100 mM Nacl Relative dry masscompared to control Gene name Mean (g) Std Error (%) Pver + 6669(negative 0.05635 0.00674 100 control) Rab7 positive control) 0.06560.00674 117.1428571 ABST_1 0.074417 0.00389 132.8875 ABST_19 0.0545670.00389 97.44107143 ABST_36 0.059117 0.00389 105.5660714

Table 16 summarises the results of the absolute and relative dry mass ofthe of the T2 transgenic lines overexpressing the ABST genes underregular growth conditions.

TABLE 16 Summary of absolute (g) and relative dry mass (relative tonegative control %) of the T2 transgenic lines overexpressing the ABSTgenes under regular growth conditions T2 plants overexpressing the ABSTgene T2 plants overexpressingthe ABST under the 35S promoter gene underthe 6669 promoter Relative Relative Mean dry expression Mean dryexpression Gene name mass (g) Std Error % mass (g) Std Error % Negativecontro1 0.048666667 0.006557 100 0.0391 0.006154 100 ABST_1 0.0564166670.007331 117.534722 0.05790333 0.006251 148.4700854 ABST_6 0.0526666670.006557 109.722222 0.0561 0.006154 143.8461538 ABST_19 0.0432 0.00655790 0.03553333 0.006251 91.11111103 ABST_22 0.066 0.006557 137.5 ND ND NDABST_36 0.049333333 0.006557 102.777778 0.0484 0.006154 124.1025641ABST_37 0.0632 0.006557 131.666667 ND ND ND

As summarized in Table 16 above, transgenic T₂ Arabidopsis plantsover-expressing ABST genes of the present invention as set forth in SEQID NOs: 1 (ABST_1) and 4 (ABST_6) showed significant elevation of drymass both under the 35S promoter and the 6669 promoter (ABST_1: 18%, 49%respectively, ABST_6: 10%, 44% respectively). SEQ ID NO: 13 (ABST_36)over-expression caused elevation in dry mass only under the regulationof 6669 promoter (24%). ABST_22 and _37 over-expression (SEQ ID NOs: 9and 14 respectively), under the regulation of the 35S promoter, causedmore than a 40% increase in the dry mass of the transgenic lines.

The results showed that all five tested genes improve vegetative growthdevelopment under favorable conditions. ABST_1 and 6 improve plant vigorin both regulation modes (6669 and 35S promoters).

The specific gene-promoter combination has a significant effect on drymass elevation.

To further examine if elevation in plant vigor has a direct effect ontotal seed weight, T₂ plant seeds (grown under regular conditions)over-expressing ABST_1, (SEQ ID NO: 1) 6 (SEQ ID NO: 4), 36 (SEQ ID NO:13) and 37 (SEQ ID NO: 14) under the 6669 promoter were weighed. Theseeds from plants over-expressing ABST_1 and 36 weighed 50% morecompared to control lines as illustrated in FIG. 7 and Table 17 below.

TABLE 17 Average seed weight of arabidopsis lines over-expressing ABSTgenes 1, 6, 19, 36, 37 under the 6669 promoter Least Sq Level of Levelof Std Level Mean significance* significance* Error ABST_1 0.062 A 0.006ABST_36 0.061 A 0.005 ABST_37 0.053 A B 0.004 ABST_6 0.048 A B 0.006Negative control 0.048 A B 0.005 Positive control-Rab7 0.043 B 0.005ABST_19 0.043 B 0.004 *Levels not connected by same letter aresignificantly different

The effect of over-expression of ABST genes in plants subjected to saltstress on dry mass was tested using the same plant populations grownunder continuous irrigation of saline water.

As summarized in Table 18 below, ABST_6 and 36 (SEQ ID NOs: 4 and 13respectively) increased the plant dry mass under both the 35S and 6669promoters (ABST_6: 25%, 16% respectively; ABST_36: 15%, 33%respectively). Plants over-expressing ABST_1 (SEQ ID NO: 1) showedhigher dry mass only under the 6669 promoter (66%). ABST_22 and 37 (SEQID NOs: 9 and 14 respectively) were tested only under the 35S promoterand showed a significant increase (>43%) of plant dry mass compared tocontrol line.

TABLE 18 Summary of absolute (g) and relative dry mass (relative tonegative control %) of the T2 transgenic lines overexpressing the ABSTgenes under salt stress conditions T2 plants overexpressing T2 plantsoverexpressing the ABST gene under the the ABST gene under the 35Spromoter 6669 promoter Relative Relative expression expression Mean dryStd compare to Mean dry Std compare to Gene name mass (g) Error control(%) mass (g) Error control (%) Negative control 0.041 0.005 100.0000.019 0.002 100.000 ABST_1 0.035 0.006 93.280 0.032 0.002 166.035 ABST_60.052 0.005 124.750 0.022 0.001 114.480 ABST_19 0.040 0.005 102.2730.017 0.002 87.753 ABST_22 0.056 0.005 142.455 ND ND ND ABST_36 0.0490.005 115.910 0.027 0.002 132.576 ABST_37 0.059 0.005 143.068 ND ND NDND = not determined

Table 19 below summarizes the results of all the dry mass and seedmeasurements that were performed on the transgenic arabidopsisover-expressing the ABST genes. (calculated as percentage comparenegative control).

TABLE 19 Dry Mass and seed measurements Growth conditionsMeasurement/Gene name ABST_1 ABST_6 ABST_22 ABST_36 ABST_37 FavorableIncrease (%) of dry mass of plant over 18 10 38 0 32 conditionsexpressing ABST genes under 35S promoter Favorable Increase (%) of drymass of plant over 49 44 ND 24 ND conditions expressing ABST genes under6669 promoter Favorable Increase of seeds weight in plants over 28 0 ND27 11 conditions expressing ABST genes under 6669 regulation Salt stressIncrease of dry mass of plant over 66 14 ND 33 ND conditions expressingABST genes under 6669 regulation Salt stress Increase of dry mass ofplant over 0 25 42 16 43 conditions expressing ABST genes under ABST 35Sregulation Table 19 continued

Example 10 Evaluating Growth of Transgenic Tomato Plants CultivatedUnder Abiotic Stress Conditions

The M82 tomato variety strain (Evoline 3) was used to determine whetherimprovement of plant vigor under stress may be translated intoimprovement of commercial yield. ABST genes 1, 6 and 36 (SEQ I.D. NOs.1, 4 and 13 respectively) were transformed under the regulation of the6669 promoter. As described in Example 9, all three gene combinationsshowed significant improvement of stress tolerance in arabidopsisplants. The genes were introduced into M82 variety by crossingtransgenic miniature tomato lines with M82 plants. To represent thevariation of the position effect, a pool of pollen from transgenic linesrepresenting 4 different insertion events from each one of the geneswere used as the male parent. The F1 hybrids were used for furtherevaluation.

The segregating F1 populations were divided into two isogenicpopulations, i.e. plants over-expressing the ABST genes of the presentinvention and plants that were not transformed to over-express ABSTgenes (NT) from the same populations used as negative controls. Duringthe first three weeks, all the plants were grown in a nursery underregular conditions. Following this period the plants were transplantedinto a commercial greenhouse under two different treatments. The firstgroup of plants was grown under favorable conditions while the secondgroup was grown under continuous irrigation of saline water (180 mMNaCl). Each transgenic line was compared to a NT plant derived from thesame F1 population. The plants were evaluated for their plant and fruitperformance at a stage where the percentage of red fruit was on average80%.

Results:

The plants over-expressing ABST genes showed improved vigor and largerroot systems than the NT plants. In addition, the plants over-expressingABST genes showed improved yield under salt stress conditions.

Specifically, plants over-expressing ABST genes showed an elevation inthe fresh weight of both canopies and roots. The populations expressingABST_1, 6 and 36 showed elevations of 165%, 162%, 206% respectively incanopy fresh weight compared to NT populations (Table 20) and higherroot weight of 162%, 168% and 121% respectively compared to NT plants.

TABLE 20 Summary of absolute (g) and relative fresh mass (relative tonegative control %) of the canopies of M82 tomato T2 transgenic linesoverexpressing the ABST genes under saline water irrigation M82 tomatoT2 plants overexpressing the ABST gene under the 6669 promoter MeanRelative canopies increase fresh Std Level of compare to Gene nameweight Error significancy* control (%) [MT] × [M82]_T 142.780 26.375 c99.986 (transgenic negative control) Plasmid backbone only) [ABST_1] ×[M82] 235.780 32.302 ab 165.112 [ABST_6] × [M82] 231.280 45.682 abc161.961 [ABST_36] × [M82] 294.168 52.263 a 206.000 *Levels not connectedby same letter are significantly different)

To further prove that this elevation in weight was due to accumulationof biomass and not only due to water accumulation, the dry mass of boththe canopies and roots was measured. The dry mass of the canopiesincreased by 156%, 145% and 161% in the lines expressing ABST_1, 6, 36respectively compared to the corresponding NT lines. Similar effectswere observed in the roots of these lines. Roots of the plantsover-expressing ABST genes contained 40% (as summarized in Table 21below) more dry matter than roots of the NT control plants (ABST_1—168%,6−159% and 36—140%).

TABLE 21 Summary of absolute (g) and relative dry mass (relative tonegative control %) of the roots of M82 tomato T2 transgenic linesoverexpressing the ABST genes under saline water irrigation M82 tomatoT2 plants overexpressing the ABST gene under the 6669 promoter Relativeincrease compare Mean root to dry Level of control Gene name weight (g)Std Error significancy* (%) [MT] × [M82]_T 2.073 0.310 b 100.000(transgenic negative control) [ABST_1] × [M82] 3.700 0.379 a 168.182[ABST_6] × [M82] 3.500 0.424 a 159.091 [ABST_36] × [M82] 3.067 0.693 ab139.394 *Levels not connected by same letter are significantlydifferent)

Only transgenic plants over expressing ABST_36 had a lower root/shootmass ratio than control plants (Table 22 below). This lower ratio (0.067compared to more than 0.08 in control plants) is the result of a nearly50% increase in shoot fresh weight rather than a major decrease in rootfresh weight. This finding suggests that under stress growth conditions,the ABST_36 over-expressing plants require a relatively lower root massto support shoot growth and development.

TABLE 22 Ratio between root to shoot mass in control plants and threetransgenic lines over expressing ABST_1, 6, 36 Gene name Ratio dryweight roots per canopy [MT] × [M82] 0.086287522 Non-transgenic negativecontrol [ABST_1] × [M82] 0.083090052 [ABST_6] × [M82] 0.085003036[ABST_36] × [M82] 0.067

Fruit yield was analyzed by measuring the number of fruit clusters, thenumber of green and red fruits and weight of the green and red fruits ineach one of the lines.

Plants over-expressing ABST_36 comprise (37%) significantly more fruitclusters compared to control line suggesting a link between vigor andyield potential as summarized in Table 23 below.

All three tested ABST genes of the present invention improved totalfruit yield as illustrated in Table 24. Specifically, ABST_1 increasedfruit yield by 137%. ABST_6 increased fruit yield by 151%. ABST_36increased fruit yield by 191%. The relative large internal variationwithin populations reflects the variation that exists between differentinsertion events.

A more detailed analysis of the yield determinants showed that most ofthe additional yield (50% to 90%) was due to elevation in the weight ofgreen fruit (Table 25). In addition most of the elevation in yield wasdue to an increase in the number of fruits rather than enlargement offruit size (Table 26).

The significant difference in the number of green fruits between thelines largely depends on the physiological status of the plants. Thecontrol NT plants wilted much earlier while the plants over-expressionABST genes continued to develop for a longer period.

TABLE 23 Average number of fruit clusters in transgenic linesover-expressing the ABST genes and control lines M82 tomato T2 plantsoverexpressing the ABST gene under the 6669 promoter Mean Relativenumber of Level of per control Gene name clusters Std Error significant(%) [MT] × [M82]_T 18.586 1.493 b 100.000 (Transgenic negative control)[ABST_1] × [M82] 19.402 1.829 ab 104.874 [ABST_6] × [M82] 22.533 2.586ab 121.799 [ABST_36] × [M82] 25.467 2.876 a 137.660

TABLE 24 Total fruit yield in transgenic lines over-expressing the ABSTgenes and control lines M82 tomato T2 plants overexpressing the ABSTgene under the 6669 promoter Mean of Relative total fruits Level of percontrol Gene name weight (g) Std Error significant (%) [MT] × [M82]_T431.974 61.573 c 100.000 (Transgenic negative control) [ABST_1] × [M82]592.724 71.693 bc 137.104 [ABST_6] × [M82] 652.338 101.389 bc 148.869[ABST_36] × [M82] 825.934 115.330 b 187.029

TABLE 25 Green fruit weight of transgenic lines over-expressing the ABSTgenes and control lines (gram) M82 tomato T2 plants overexpressing theABST gene under the 6669 promoter Mean of Relative green fruits Level ofper control Gene name weight (g) Std Error significant (%) [MT] ×[M82]_T 145.401 47.168 c 100.000 (Transgenic negative control) [ABST_1]× [M82] 221.722 55.219 bc 152.491 [ABST_6] × [M82] 256.431 78.091 bc176.362 [ABST_36] × [M82] 421.925 87.206 b 290.182

TABLE 26 Average number of the green fruits that were produced in thetransgenic lines over-expressing the ABST genes and control lines M82tomato T2 plants overexpressing the ABST gene under the 6669 promoterMean of Relative number of Level of per control Gene name green fruitsStd Error significant (%) [MT] × [M82]_T 26.127 6.173 bc 100.000(Transgenic negative control) [ABST_1] × [M82] 41.666 7.146 ab 160.254[ABST_6] × [M82] 41.047 10.106 ab 157.875 [ABST_36] × [M82] 58.70311.756 a 225.780

FIGS. 8A-8D depict control and transgenic plants of the presentinvention illustrating the increase in yield following over-expressionof the ABST genes of the present invention.

Comparison between lines expressing ABST_1, 6, 36 under favorableconditions to control lines under favorable conditions did not show anysignificant changes in the vigor of the vegetative parts or in the fruityield. The following parameters were tested: fresh and dry weight of theroots and canopies, green fruit weight, red fruit weight and the averagegreen and red fruit diameter. In all these parameters no differenceswere detected.

Table 27 below summarizes the results on crop yield followingover-expression of ABST in tomato plants.

TABLE 27 Comparison of plant and fruit performances between transgenicand control plants grown under favorable and salt stress conditions(irrigation of 180 Mm NaCl) Control ABST_1 ABST_6 ABST_36 Control ABST_1ABST_6 ABST_36 (180 mM (180 mM (180 mM (180 mM (Water) (Water) (Water)(Water) NaCl) NaCl) NaCl) NaCl) Canopy ND ND ND ND 143 c 236 ab 231 ab295 a fresh weight Root  84 a  74 a  72 a  87 a  16 c 27 a 28 a  20 abfresh weight Number  195 a  185 a  186 a  171 a  66 c  90 bc  87 bc 104b of fruits per plant Total 2750 a 2845 a 2846 a 2501 a 432 c 593 bc 652bc 826 b Fruit weight per plant

Tomato plants that were crossed between transgenic miniature tomatoesand the M82 line (line 3) were also grown under both salt stress andfavorable conditions. The salt stress was carried out by continuousirrigation of saline water containing 180 mM NaCl.

The miniature populations expressing ABST_1 and 22 showed the highestimprovement in salt tolerance compare to the control line based on plantand root mass and yield performance. The dry mass of the transgeniclines expressing ABST_1, 22 was increased by 300% and 257% respectively.ABST_36 improved both shoot mass and yield by about 30% as compared tocontrol plants. The lines expressing ABST_1 and 22 showed the highestyield performance (165% and 140% respectively) compared to controlpopulation.

ABST_36 and 37 were also over-expressed in a line 2 tolerant tomatoplant (Y361) and its expression was compared to that in a line 1sensitive tomato plant (Shirly). As illustrated in FIGS. 9A-9C, ABST_36expression was higher in the tolerant plants in both roots and leaves.ABST_37 was up-regulated in leaves of both tolerant and sensitive lines.However in the tolerant line (line 2) the expression was up-regulated inleaves much faster than in leaves of sensitive line (line 1).Up-regulation in roots occurred only in the tolerant lines (line 2).

Hence, the results from Examples 1-10, clearly indicate that the abioticstress tolerance genes of the present invention described herein can bereadily isolated and utilized to substantially increase tolerance toabiotic stress and/or biomass in plants.

Example 11 Identifying Putative Abiotic Stress-Tolerance Genes fromMonocots

Monocot ortholog sequences for the 5 putative ABST tomato genes (SEQI.D. NOs. 1,4, 9, 13 and 14) were sought. Monocot genomic databasesnamely NCBI (Hypertext Transfer Protocol://World Wide Web (dot) ncbi(dot) nlm (dot) nih (dot) gov/) and TIGR (Hypertext TransferProtocol://World Wide Web (dot) tigr (dot) org/) databases of Maize,Sorghum and Barley were initially screened. The expressed sequence tags(ESTs) and cDNA sequences were clustered and assembled using the LEADS™software (Compugen) and compared to the TIGR (Hypertext TransferProtocol://World Wide Web (dot) tigr (dot) org/) databases of the abovemonocots. Overall, clustering of 372,000 maize ESTs resulted in 41,990clusters among them 19,870 singletons. In Sorghum, about 190,000 ESTswere clustered into 39,000 clusters while in barley 370,500 ESTsgenerated 50,000 different clusters each representing a different gene.A digital expression profile summary was compiled for each clusteraccording to all keywords included in the sequence records comprisingthe cluster.

Yet, while comparing the monocot sequences to the tomato ABST genes,sequence homology levels differed dramatically, ranging from 45% to 88%.Moreover, the in-silico expression profile of the monocot genes did notalways fit the profile of a gene involved in ABS tolerance.

In an attempt to identify the best orthologues for the tomato ABST genesvarious additional factors were analyzed. First, the sequences of the 5tomato ABST genes (SEQ ID NO: 1, 4, 9, 13 and 14) and their deducedpolypeptide sequences (SEQ ID NOs: 236-240) were compared to all monocotputative proteins, encoded by DNA sequences of gene clusters mentionedabove. The comparison was performed on the protein level looking foridentity higher than 45% along the entire protein sequences. Table 28shows the best homologous genes and their identity level to the tomatoABST proteins. Next, these monocot proteins originating from differentmonocot species (barley, sorghum and maize) were screened based on theirexpression pattern during the development of several monocot species.This screening was based on digital expression of the genes, asdescribed above. The genes were selected based on three criteria: geneswith higher expression in roots, roots and leaves and or induceexpression by treatments representing soil stress conditions (drought,salinity, soil deficiencies). The increase of expression was onlycounted in cases where the increase was greater than 2 fold (relative tothe random EST distribution) with significance probability lower than0.05. Table 29 summarizes the expression profile of the genes indifferent organ or tissues and the treatments that set off significantelevation in their expression level.

TABLE 28 The level of homology between the tomato ABST genes and theirhomologs from monocots % Identity (Percenrtage from TIGR Name/Acc Levelof the entire Tomato gene No of homology protein SEQ ID NO Homologousgene Plant origin (e value) sequence) 1 TC104838 Sorghum 2E−70 88% SEQID NO: 93 TC103857 Sorghum 2E−70 88% TC258871 Maize 1E−69 86% TC139195Barley 5E−69 86% 4 TC94284 Sorghum 3E−43 45% SEQ ID NO: 94 TC132394Barley 6E−40 44% 9 TC93449 Sorghum 1E−99 58% SEQ ID NO: 95 TC146720Barley 3E−99 58% 13 TC92953 Sorghum 7E−59 47% SEQ ID NO: 96 TC91426Sorghum 4E−98 74% SEQ ID NO: 97 TC91474 Sorghum 5E−98 72% TC263205 Maize2E−97 74% 14 TC103772 Sorghum 1E−52 49% SEQ ID NO: 98 TC148356 Barley1E−54 46% TC260731 Maize 1E−54 46%

TABLE 29 The expression profile of ABST homologous in silico genes asrepresented by statistical analysis of their EST distribution Foldincrease (All results are Fold increase Name of Organs/tissuessingnificant Treatments that (all results are Homologous with thehighest in P value induce th singnificant in gene Plant species geneexpression >0.05) expression level P value >0.05) TC104838 SorghumPollen preanthesis 3 Ethylene, 2 SEQ ID NO: 93 stage drought TC103857Sorghum Diverse 2 None* None* expression TC258871 Maize Diverse 2 None*None* expression, preferentially in cell lignification region of leavesTC139195 Barley In various grain 2-3.5 None None tissues TC94284 SorghumLeaves, 4.5 Drought, 4 SEQ ID NO: 94 roots during fruit 2 nitrogen 2loading deficiencies, 2 soil acidity TC132394 Barley Leaves, coleoptile2.5 None None mainly during 3 fruit development TC93449 Sorghum Flowersovary 3 Salinity stress 4 SEQ ID NO: 95 TC146720 Barley Seeds 2 Coldstress, 3 preferentially in Fusarium 3.5 the embryo and infectionscutellum during ripening TC92953 Sorghum Leaves during 2 Drought, 4 SEQID NO: 96 fruit loading Nitrogen- 4 deficiency, 2.5 salinity (150 Mm)TC91426 Sorghum Young roots 12 Ethylene, 4 SEQ ID NO: 97 etiolation,soil 3 acidity 12 TC91474 Sorghum Entire seedling 2 Etiolation 16TC263205 Maize Primary root 3 Drought 2 system in seedling stageTC103772 Sorghum Young roots 2 Drought, 2 SEQ ID NO: 98 soil acidity 2TC148356 Barley Callus, leaves in 4, 2 Infection by 2 the vetatativeBlumeria stage graminis TC260731 Maize Root preferntialy 2.5 None Noneprimary roots None*—None of the treatments with significant elevation indigital expression could be considered as soil stress treatment

A combination of the above screening as described in Table 28 and inTable 29 revealed the final list of six monocot genes that are predictedto be the most related to the tomato ABST genes (SEQ ID NOs. 93, 94, 95,96, 97 and 98).

Another type of sequence alignment for finding putative orthologoussequences from barley, rice, maize and sorghum, using the tomato ABSTgenes as involved the use of an evology system. Digital expressionanalysis was performed on these genes allowing for the identification ofother putative monocot orthologs. The results were corroborated byphylogenetic analysis which studies the relationships between the tomatoABST genes and the putative monocot orthologs.

The Evology system is a method for constructing ortholog groups acrossmultiple eukaryotic taxa, using the Markov cluster algorithm to groupputative orthologs and paralogs. The method coherent with the groupsidentified by EGO (Hypertext Transfer Protocol://World Wide Web (dot)tigr (dot) org/tdb/tgi/ego/index (dot) shtml) but improved theidentification of “recent paralogs” since EGO is easily misled by thefunctional redundancy of multiple paralogs and by the absence of trueorthologs within incomplete genome data set as in most of the plantspecies.

The Evologs is a tool for large-scale automated eukaryotic orthologgroup identification. To resolve the many-to-many orthologousrelationships inherent in comparisons across multiple genomes, Evologsapplied the Markov Cluster algorithm (Hypertext TransferProtocol://micans (dot) org/mcl/), which is based on probability andgraph flow theory and allows simultaneous classification of globalrelationships in a similarity space. MCL simulates random walks on agraph using Markov matrices to determine the transition probabilitiesamong nodes of the graph. The MCL algorithm has previously beenexploited for clustering a large set of protein sequences, where it wasfound to be very fast and reliable in dealing with complicated domainstructures. Evologs generates clusters of at least two proteins, whereeach cluster consists of orthologs or paralogs from at least onespecies.

The putative orthologs were obtained using three levels of stringency.The first group with the lowest level (p value<=1e-20 andidentity>=50%), the second group with moderate level of stringency (pvalue<=1e-42 and identity>=50%) and the third group with the higheststringency include p value<=1e-70 and identity>=70%.

-   1. Eight genes were identified as putative orthologs for ABST_1.    This group was defined using the highest stringency parameters    (highest cutoff—level 3).-   2. Nine monocot genes were identified as ABST_6 putative orthologs.    These genes were found only after filtering under the lowest    stringency alignment (level 1) parameters. This reduces the    probability of finding a real monocot ortholog.-   3. Eight monocot genes were identified as ABST_22 putative    orthologs. These genes were found by using the highest stringency    parameters (highest cutoff—level 3).-   4. Twenty three putative ortholog genes were found for ABST_36    (Table 2). This group was found by using the highest stringency    parameters (the highest cutoff—level 3).-   5. Fourteen putative orthologs for ABST_37 were found only after    reducing the alignment parameters to the second stringency level.    However since the genes are transcription factors more accurate    comparison should be done on their binding domains.

These genes were subjected to digital expression analysis. Genes thatwere identified as being up-regulated under stress conditions underwentphylogenetic analysis. The phylogenetic trees showed similar distancesbetween tomato, Arabidopsis and monocots supporting the claim thatconservation in function in Arabidopsis and tomato strongly indicatesconservation in function in monocot (data not shown).

A final list of ten candidate monocot ortholog genes was drawn up, asdetailed in Table 30 below.

TABLE 30 List of ten candidate monocot orthologues as revealed by evologanalysis, phylogenetic analysis and digital expression analysis %Identity TIGR (Percentage from the Tomato gene Name/Acc No of entireprotein SEQ ID NO: Homologous gene Plant origin sequence) 1 TC104838Sorghum 88% SEQ ID NO: 93 4 TC94284 Sorghum 45% SEQ ID NO: 94 9 TC93449Sorghum 58% SEQ ID NO: 95 TC102291 Sorghum 54% SEQ ID NO: 241 13TC131030 Barley 72% SEQ ID NO: 242* AF057183 maize 70% SEQ ID NO: 245TC249365 maize 70% SEQ ID NO: 244 TC249366 maize 70% SEQ ID NO: 243TC263205 Maize 74% SEQ ID NO: 246 14 TC103772 Sorghum 49% SEQ ID NO: 98*SEQ ID NO: 242 is identical to SEQ ID NO: 74

The digital expression profile for TC104838 (SEQ ID NO:93) is listed inTables 31-33 herein below.

TABLE 31 Expression of TC104838 in different anatomical regions of theplant ESTs in  ESTs in Expected Keyword Gene Production ESTs Foldp-value flower 3 26937 1.11986 2.6789 0.0865946 pollen 3 8840 1 30.00431486 leaf 1 17487 1 1 0.535872 seedling 2 95402 3.96618 0.5042630.970844

TABLE 32 Expression of TC104838 during development ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value flowering 4 327051.35966 2.94192 0.0301425 8-14 days pre- 3 8840 1 3 0.00431486 anthesispost-flowering 1 5768 1 1 0.216515 germination 2 104379 4.33939 0.4608950.985772 1.5 week 2 47911 1.99182 1.00411 0.636908 vegetative 1 21465 11 0.615042 5 weeks old 1 9746 1 1 0.34123

TABLE 33 Expression of TC104838 under various treatments ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 2 18855 1 20.180136 drought stress 1 5768 1 1 0.216515 after flowering droughtstress, 1 9746 1 1 0.34123 7, 8 days after water witheld hormone 2 260471.08286 1.84696 0.29656 treatment ethylene- 2 6261 1 2 0.0256292 inducedwith ACC, 27 and 72 hours after induction

The digital expression profile for TC94284 (SEQ ID NO:94) is listed inTables 34-36 herein below.

TABLE 34 Expression of TC94284 in different anatomical regions of theplant ESTs in ESTs in Expected Keyword Gene Production ESTs Fold p-valueleaf 5 17487 1.14242 4.37668 0.0032544 seedling 6 95402 6.23257 0.9626840.675068 leaf 2 19738 1.28947 1.55102 0.375678 root 2 7258 1 2 0.078916

TABLE 35 Expression of TC94284 during development ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value flowering 4 327052.1366 1.87213 0.148602 post- 4 5768 1 4 0.000374052 floweringgermination 6 104379 6.81904 0.87989 0.795559 1.5 week 2 47911 3.130010.638976 0.864874 2 weeks old 4 27953 1.82616 2.19039 0.094453vegetative 1 21465 1.4023 0.713114 0.7769 4 weeks old 1 8221 1 10.423423

TABLE 36 Expression of TC94284 under various treatments ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 4 188551.23179 3.24731 0.0271328 drought stress 4 5768 1 4 0.000374052 afterflowering nutrient 2 9927 1 2 0.134282 deficiencies Nitrogen 2 3313 1 20.0189181 deficient pathogen 3 17272 1.12837 2.6587 0.0951298

The digital expression profile for TC93449 (SEQ ID NO:95) is listed inTables 37-39 herein below.

TABLE 37 Expression of TC93449 in different anatomical regions of theplant ESTs in ESTs in Expected Keyword Gene Production ESTs Fold p-valuecallus 2 9585 1.36622 1.46389 0.40017 cell 2 9585 1.36622 1.463890.40017 suspension flower 6 26937 3.83953 1.56269 0.17419 ovary 4 94341.3447 2.97465 0.0426186 leaf 3 17487 2.49255 1.20359 0.461176 seedling13 95402 13.5983 0.955999 0.676721 leaf 1 19738 2.8134 0.355442 0.949846root + leaf 5 19261 2.74541 1.82122 0.131853 callus 2 9585 1.366221.46389 0.40017

TABLE 38 Expression of TC93449 during development ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value flowering 7 327054.66168 1.5016 0.169309 8 weeks old 4 9434 1.3447 2.97465 0.0426186(immature) post-flowering 1 5768 1 1 0.56683 pre-anthesis 2 8663 1.23481.6197 0.352104 germination 13 104379 14.8779 0.873779 0.841438 1 week 119538 2.78489 0.35908 0.948201 1.5 week 11 47911 6.8291 1.610750.0526309 2 weeks old 1 27953 3.98435 0.250982 0.987187 vegetative 221465 3.05956 0.653688 0.82924 4 weeks old 2 8221 1.1718 1.706780.328675

TABLE 39 Expression of TC93449 under various treatments ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 1 188552.68754 0.372087 0.942184 drought stress 1 5768 1 1 0.56683 afterflowering heat stress 1 8875 1.26502 0.790502 0.727378 4 and 24 hours 18875 1.26502 0.790502 0.727378 at 40-42° C. hormone 6 26047 3.712671.61609 0.155303 treatment ethylene- 4 6261 1 4 0.0111844 induced withACC, 27 and 72 hours after induction Salicylic acid- 2 4793 1 2 0.148347treated nutrient 1 9927 1.41497 0.70673 0.767414 deficiencies Irondeficient 1 3353 1 1 0.382937 pathogen 3 17272 2.4619 1.21857 0.452792Resistant 1 9051 1.29011 0.775131 0.734507 plants, 48 h afterColletotrichum graminicola innoculation (fungi) Susceptible 2 82211.1718 1.70678 0.328675 plants, 48 h after Colletotrichum graminicolainnoculation (fungi) salinity 4 6080 1 4 0.010119 150 mM NaCl 4 6080 1 40.010119 for 3, 6, 12 and 24 hr

The digital expression profile for TC102291 (SEQ ID NO: 241 and 247) islisted in Tables 40-42 herein below.

TABLE 40 Expression of TC102291 in different anatomical regions of theplant ESTs in ESTs in Expected Keyword Gene Production ESTs Fold p-valuecallus 4 9585 1.48007 2.70257 0.0576229 cell 4 9585 1.48007 2.702570.0576229 suspension leaf 5 17487 2.70026 1.85167 0.126138 seedling 1495402 14.7315 0.950342 0.688991 leaf 2 19738 3.04785 0.6562 0.825957root + leaf 9 19261 2.97419 3.02603 0.00168405

TABLE 41 Expression of TC102291 during development ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value flowering 3 327055.05016 0.594041 0.904756 post-flowering 3 5768 1 3 0.0581305germination 14 104379 16.1177 0.868609 0.854646 1 week 2 19538 3.016970.662917 0.821366 1.5 week 4 47911 7.3982 0.540672 0.962528 2 weeks old8 27953 4.31637 1.85341 0.0543811 vegetative 5 21465 3.31453 1.508510.230885 5 weeks old 3 9746 1.50493 1.99344 0.188518 pre-flowering 23341 1 2 0.0935317

TABLE 42 Expression of TC102291 under various treatments ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 8 188552.9115 2.74772 0.00599968 drought stress 3 5768 1 3 0.0581305 afterflowering drought stress 2 3341 1 2 0.0935317 before flowering droughtstress, 3 9746 1.50493 1.99344 0.188518 7, 8 days after water witheldheat stress 1 8875 1.37044 0.729694 0.755364 4 and 24 hours 1 88751.37044 0.729694 0.755364 at 40-42° C. hormone 6 26047 4.02206 1.491770.204473 treatment Abscisic acid- 5 4306 1 5 0.000458231 treatedethylene- 1 6261 1 1 0.626674 induced with ACC, 27 and 72 hours afterinduction light response 1 18685 2.88525 0.34659 0.953042 etiolated 110663 1.64653 0.607337 0.817519 nutrient 1 9927 1.53288 0.6523660.794035 deficiencies Nitrogen 1 3313 1 1 0.403524 deficient pathogen 217272 2.66706 0.749889 0.761847 Resistant 2 9051 1.39761 1.431010.411127 plants, 48 h after Colletotrichum graminicola innoculation(fungi) salinity 3 6080 1 3 0.0659796 150 mM NaCl 3 6080 1 3 0.0659796for 3, 6, 12 and 24 hr

The digital expression profile for TC131030 (SEQ ID NO:242 and 248) islisted in Tables 43-45 herein below.

TABLE 43 Expression of TC131030 in different anatomical regions of theplant ESTs in ESTs in Expected Keyword Gene Production ESTs Fold p-valueroot 5 16560 1 5 1.48052E−06 seedling 1 57039 1 1 0.662622 root 1 1988 11 0.0341428 shoot 1 8317 1 1 0.136442

TABLE 44 Expression of TC131030 during development ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value germination 1 927781.61655 0.6186 0.847952 5-8 days 1 29670 1 1 0.417607 seedling 5 33561 15 4.84613E−05 3 weeks old 5 21898 1 5 5.90628E−06

TABLE 45 Expression of TC131030 under various treatments ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 3 8410 1 30.00027555 drought 2 4939 1 2 0.00296904 stressed drought 1 1496 1 10.0257848 stressed (6 and 10 h on moist paper in light) light response 26815 1 2 0.00557109 etiolated 1 4697 1 1 0.0791 Low light 1 888 1 10.0153731 waterlogged 1 2259 1 1 0.0387209 waterlogged 1 2259 1 10.0387209

The digital expression profile for TC249366 (SEQ ID NO:243 and 251) islisted in Tables 46-48 herein below.

TABLE 46 Expression of TC249366 in different anatomical regions of theplant ESTs in ESTs in Expected Keyword Gene Production ESTs Fold p-valueroot 26 36059 2.69511 9.6471 2.22045E−15 primary root 26 33886 2.532710.2657 0 system

TABLE 47 Expression of TC249366 during development ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value germination 26 748695.59584 4.64631 7.54952E−15 young 26 34586 2.58502 10.058 0 seedling

TABLE 48 Expression of TC249366 under various treatments ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 21 212161.58572 13.2432 7.77156E−16 CONTROL 8 5966 1 8 1.00075E−08 well watered0 h water stress 2 6113 1 2 0.0761554 48 h water stress 7 6417 1 73.8251E−07 5 h water stress 4 2720 1 4 4.97583E−05 5 h and 48 h,Subtracted library

The digital expression profile for TC249365 (SEQ ID NO:244 and 250) islisted in Tables 49-51 herein below.

TABLE 49 Expression of TC249365 in different anatomical regions of theplant ESTs in ESTs in Expected Keyword Gene Production ESTs Fold p-valueflower 11 89278 11.7349 0.937375 0.649792 seedling + 8 9012 1.184566.75357 2.20576E−05 female flower silk 3 1536 1 3 0.001119 leaf 3 356894.69104 0.639517 0.859653 mix 19 90046 11.8359 1.60529 0.0168681 root 1136059 4.73968 2.32083 0.00638112 primary 11 33886 4.45405 2.469660.00399824 root system seedling 12 32466 4.26741 2.81201 0.000841861seedling + 8 9012 1.18456 6.75357 2.20576E−05 female flower shoot 416152 2.12306 1.88408 0.162096

TABLE 50 Expression of TC249365 during development ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value flowering 11 8927811.7349 0.937375 0.649792 developed 8 9012 1.18456 6.75357 2.20576E−05seedling + silking silking 3 2160 1 3 0.00293981 germination 26 748699.84095 2.64202 3.92859E−07 developed 9 31271 4.11033 2.1896 0.0196511seedling developed 8 9012 1.18456 6.75357 2.20576E−05 seedling + silkingyoung 9 34586 4.54606 1.97974 0.034885 seedling mix 19 70970 9.328462.03678 0.00110629

TABLE 51 Expression of TC249365 under various treatments ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 7 212162.78868 2.51015 0.0204171 CONTROL 1 5966 1 1 0.546304 well watered 0 hwater stress 1 6113 1 1 0.555122 48 h water stress 5 h 5 6417 1 50.00154268 mix 3 36475 4.79436 0.625736 0.869757 pathogen 3 2260 1 30.00333671 Fusarium, 6 h 3 667 1 3 9.9034E−05 post infection salinity 43579 1 4 0.00127889 150 mM NaCl 4 3579 1 4 0.00127889 24 h

The digital expression profile for AFO57183 (SEQ ID NO:245 and 249) islisted in Tables 52-54 herein below.

TABLE 52 Expression of AF057183 in different anatomical regions of theplant ESTs in ESTs in Expected Keyword Gene Production ESTs Fold p-valueflower 15 83646 25.2528 0.593995 0.994869 pollen 1 11265 3.400910.294039 0.968401 seedling + 9 8119 2.45113 3.67178 0.000831679 femaleflower silk 5 1651 1 5 0.000156853 leaf 3 34159 10.3126 0.2909060.998479 mix 31 88820 26.8148 1.15608 0.205053 root 46 35521 10.72384.28952 3.55271E−15 primary root 46 33407 10.0856 4.56097 4.88498E−15system seedling 13 29180 8.80945 1.47569 0.10175 seedling + 9 81192.45113 3.67178 0.000831679 female flower shoot 4 14803 4.46903 0.8950490.65764

TABLE 53 Expression of AF057183 during development ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value flowering 15 8364625.2528 0.593995 0.994869 developed 9 8119 2.45113 3.67178 0.000831679seedling + silking silking 5 2284 1 5 0.000684783 tasseling 1 172005.19269 0.192579 0.995102 germination 62 70016 21.1379 2.93313 0developed 13 28013 8.45713 1.53716 0.0800076 seedling developed 9 81192.45113 3.67178 0.000831679 seedling + silking young 40 33884 10.22963.91023 1.82077E−14 seedling mix 30 66869 20.1878 1.48605 0.0138371

TABLE 54 Expression of AF057183 under various treatments ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 33 212416.41266 5.14607 1.42109E−14 CONTROL 11 5813 1.75495 6.268 1.69797E−06well watered 0 h water stress 3 6130 1.85065 1.62105 0.282624 48 h waterstress 13 6304 1.90318 6.83067 6.879E−08 5 h water stress 6 2825 1 60.000233093 5 h and 48 h, Subtracted library mix 6 30831 9.307890.644614 0.91149 pathogen 5 2415 1 5 0.000877511 Fusarium, 4 710 1 47.00937E−05 6 h post infection Fusarium, 1 251 1 1 0.0730116 72 h postinfection salinity 8 3603 1.08775 7.35465 1.52409E−05 150 mM 8 36031.08775 7.35465 1.52409E−05 NaCl 24 h

The digital expression profile for TC263205 (SEQ ID NO:246 and 252) islisted in Tables 55-57 herein below.

TABLE 55 Expression of TC263205 in different anatomical regions of theplant ESTs in ESTs in Expected Keyword Gene Production ESTs Fold p-valueflower 1 89278 2.53106 0.395092 0.943664 seedling + 1 9012 1 1 0.227799female flower mix 3 90046 2.55283 1.17517 0.488081 root 3 36059 1.022282.93461 0.075106 primary root 3 33886 1 3 0.0645285 system seedling 132466 1 1 0.617579 seedling + 1 9012 1 1 0.227799 female flower

TABLE 56 Expression of TC263205 during development ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value flowering 1 892782.53106 0.395092 flowering developed 1 9012 1 1 developed seedling +seedling + silking silking germination 4 74869 2.12256 1.88452germination developed 1 9012 1 1 developed seedling + seedling + silkingsilking young 3 34586 1 3 young seedling seedling mix 3 70970 2.012021.49104 mix

TABLE 57 Expression of TC263205 under various treatments ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 3 21216 1 30.0193621 water stress 5 h 1 6417 1 1 0.167604 water stress 5 h 2 2720 12 0.00259071 and 48 h, Subtracted library

The digital expression profile for TC103772 (SEQ ID NO:98) is listed inTables 58-60 herein below.

TABLE 58 Expression of TC103772 in different anatomical regions of theplant ESTs in ESTs in Expected Keyword Gene Production ESTs Fold p-valueleaf 2 17487 1.24628 1.60478 0.358705 seedling 10 95402 6.79917 1.470770.053411 leaf 2 19738 1.4067 1.42177 0.419124 leaf + root 2 9479 1 20.143927 root 2 7258 1 2 0.092071

TABLE 59 Expression of TC103772 during development ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value flowering 2 327052.33084 0.858059 0.708441 post-flowering 2 5768 1 2 0.0616601germination 10 104379 7.43895 1.34428 0.106822 1.5 week 5 47911 3.414551.46432 0.236642 2 weeks old 5 27953 1.99217 2.50982 0.0357911

TABLE 60 Expression of TC103772 under various treatments ESTs in ESTs inExpected Keyword Gene Production ESTs Fold p-value drought 2 188551.34377 1.48835 0.395639 drought stress 2 5768 1 2 0.0616601 afterflowering light response 3 18685 1.33166 2.25284 0.140391 CONTROL 3 80221 3 0.0172009 for etiolated nutrient 1 9927 1 1 0.517716 deficienciesIron deficient 1 3353 1 1 0.214459 oxidative stress 2 9479 1 2 0.1439273 12 and 27 h 2 9479 1 2 0.143927 with hydrogen peroxide and Paraquatpathogen 2 17272 1.23095 1.62476 0.352853 Resistant plants, 2 9051 1 20.133444 48 h after Colletotrichum graminicola innoculation (fungi) soilacidity 2 7258 1 2 0.092071 acid and 2 7258 1 2 0.092071 alkaline stress

Selected polynucleotide sequences (SEQ ID NOs: 93-98) were analyzed forpresence of ORFs using Vector NTI suite (InforMax, U.K.) version 6(Hasting Software, Inc: World Wide Web (dot) generunner (dot) com/).ORFs identified in each of these polynucleotide sequences were comparedto Genbank database sequences, using Blast (World Wide Web (dot) ncbi(dot) nlm (dot) nih (dot) gov/BLAST/); the ORF displaying the highesthomology to a GenBank sequence or sequences, was mapped in order toidentify an ATG start codon. The position of the ATG start codon of thisORF was then compared with that of the identified polynucleotidesequence in order to verify that each of the five sequences describedherein includes a full length ORF and an ATG start codon (thus qualifiesas a “putative monocot ABST gene”).

Polypeptides with significant homology to monocot ABST genes (SEQ IDNOs: 93-98) have been identified from the NCBI databases using BLASTsoftware (Table 61).

TABLE 61 ABST homologs ABST Polypeptide Monocot Homologue, ABST Homologyin ABST encoded by Polypeptide Polypeptide Putative Gene TIGR AcessionHomologue sequence SEQ ID NO. No Source Organism SEQ ID NO: (%) 93TC270110 Zea mays 105 100 93 TC56855 Saccharum officinarum 106 100 93TC104838 sorghum 107 100 93 TC57929 Saccharum officinarum 108 98 93TC103857 sorghum 109 98 93 TC262554 Oryza sativa 110 98 93 TC258871 Zeamays 111 97 93 TC139195 Hordeum vulgare 112 96 93 TC262556 Oryza sativa113 95 93 TC232174 Triticum aestivum 114 95 93 TC232139 Triticumaestivum 115 95 93 TC139194 Hordeum vulgare 116 95 93 CA486561 Triticumaestivum 117 100 93 TC258873 Zea mays 118 100 93 CA187014 Saccharumofficinarum 119 90 93 TC233455 Triticum aestivum 120 96 93 CF063450 Zeamays 121 98 93 CA617041 Triticum aestivum 122 100 94 TC94284 sorghum 123100 94 TC49791 Saccharum officinarum 124 95 95 TC93449 sorghum 125 10095 TC49718 Saccharum officinarum 126 95 95 TC49720 Saccharum officinarum127 96 96 TC92953 sorghum 128 100 96 TC66617 Saccharum officinarum 12990 96 TC273860 Zea mays 130 91 96 TC253191 Zea mays 131 90 98 TC103772sorghum 132 100 98 TC272084 Zea mays 133 92 98 TC60928 Saccharumofficinarum 134 94 93 TC5422 canola 135 88 93 TC904 canola 136 88 93TC121774 Solanum tuberosum 137 88 93 TC40342 Gossypium 138 88 93 TC40115Gossypium 139 88 93 TC155918 Lycopersicon 140 88 esculentum 93 TC154398Lycopersicon 141 88 esculentum 93 TC154397 Lycopersicon 142 88esculentum 93 TC153989 Lycopersicon 143 88 esculentum 93 TC120511Solanum tuberosum 144 88 93 TC113582 Solanum tuberosum 145 88 93TC112701 Solanum tuberosum 146 88 93 TC111912 Solanum tuberosum 147 8893 TC4674 Capsicum annum 148 88 93 TC270923 arabidopsis *149 87 93CD823817 canola 150 86 93 TC526 canola 151 86 93 TC525 canola 152 86 93BG442528 Gossypium 153 87 93 TC33702 Gossypium 154 87 93 TC32714Gossypium 155 87 93 TC270782 arabidopsis **156 87 93 TC225449 Glycinemax ***157 87 93 TC5255 Capsicum annum 158 88 93 TC28221 populus 159 8493 TC108140 medicago 160 85 93 TC28222 populus 161 84 93 TC94402medicago 162 84 93 TC28223 populus 163 83 93 TC102506 medicago 164 85 93TC132070 Hordeum vulgare 165 79 93 TC251944 Triticum aestivum 166 77 93NP890576 Oryza sativa 167 76 93 TC280376 Oryza sativa 168 73 93 CN009841Triticum aestivum 169 75 93 BI948270 Hordeum vulgare 170 75 93 TC259334arabidopsis 171 75 93 BQ767154 Hordeum vulgare 172 73 93 TC60345Saccharum officinarum 173 73 93 TC138474 Hordeum vulgare 174 85 93TC41472 populus 175 72 93 BJ458177 Hordeum vulgare 176 72 93 CB674176Oryza sativa 177 82 93 TC216405 Glycine max 178 88 93 AJ777371 populus179 70 93 CV019213 tobacco 180 85 93 CK215690 Triticum aestivum 181 8093 CD830784 canola 182 85 93 CA624722 Triticum aestivum 183 85 93TC32906 populus 184 76 93 CR285127 Oryza sativa 185 89 93 TC251945Triticum aestivum 186 72 94 TC274823 Oryza sativa 187 77 94 TC132394Hordeum vulgare 188 75 94 TC267180 Triticum aestivum 189 77 94 TC261921Zea mays 190 87 94 TC267181 Triticum aestivum 191 74 94 TC261922 Zeamays 192 81 94 TC267182 Triticum aestivum 193 73 95 TC249531 Zea mays194 86 95 TC232170 Triticum aestivum 195 85 95 TC146720 Hordeum vulgare196 85 95 TC249329 Oryza sativa 197 84 95 TC249532 Zea mays 198 88 95TC232150 Triticum aestivum 199 85 95 TC249330 Oryza sativa 200 76 95CB672603 Oryza sativa 201 71 95 TC32440 Gossypium 202 81 95 TC119105Solanum tuberosum 203 72 96 TC247999 Triticum aestivum 204 78 96TC247359 Triticum aestivum 205 77 96 TC132566 Hordeum vulgare 206 77 96TC248676 Triticum aestivum 207 74 96 TC249667 Oryza sativa 208 77 96TC66618 Saccharum officinarum 209 88 97 TC253495 Oryza sativa 214 90 97TC224823 Glycine max 215 75 97 TC234990 Triticum aestivum 216 74 97TC266178 Triticum aestivum 217 73 97 TC119051 Solanum tuberosum 218 8397 TC56409 Saccharum officinarum 219 75 97 TC35873 Populus 220 80 97TC119052 Solanum tuberosum 221 82 97 TC204518 Glycine max 222 85 97TC112169 Solanum tuberosum 223 84 97 TC254696 Zea mays 224 79 97TC254696 Zea mays 225 82 97 TC248906 Oryza sativa 226 77 97 TC154007Lycopersicon 227 82 esculentum 97 TC6466 Capsicum annuum 228 74 97TC131227 Hordeum vulgare 229 74 97 TC27564 Gossypium 230 71 98 TC275473Oryza sativa 210 78 98 TC267485 Triticum aestivum 211 77 98 TC148621Hordeum vulgare 212 76 98 TC275474 Oryza sativa 213 85 *SEQ ID NO: 149is identical to SEQ ID NO: 41 **SEQ ID NO: 156 is identical to SEQ IDNO: 42 ***SEQ ID NO: 157 is identical to SEQ ID NO: 40

Example 12 Generating Putative Monocot ABST Genes

DNA sequences of six putative Monocot ABST genes were synthesized byGeneArt (Hypertext Transfer Protocol://World Wide Web (dot) geneart(dot) com/). Synthetic DNA was designed in silico, based on the encodedamino-acid sequences of the Monocot ABST genes (SEQ ID NOs: 99, 100,101, 102, 103 and 104), and by using plant-based codon-usage. Thesynthetic sequences and the plant native orthologues were compared. Atleast 1 mutation per 20 nucleotide base pairs was added to avoidpossible silencing, when over-expressing the gene in favorable monocotspecies, such as maize. The planned sequences were bordered with thefollowing restriction enzymes sites polylinker—SalI, XbaI, BamHI, SmaIat the 5′ end and SacI at the 3′ end. The sequences were cloned indouble strand, PCR Script plasmid (GeneArt).

Example 13 Cloning the Putative ABST Genes

The PCR Script plasmids harboring the synthetic, monocot-based ABSTgenes were digested with the restriction endonucleases XbaI and SacI(Roche), purified using PCR Purification Kit (Qiagen, Germany), andinserted via DNA ligation using T4 DNA ligase enzyme (Roche) andaccording to the manufacturer's instructions, into pKG(NOSter), (SEQ IDNO: 233) and pKG(35S+NOSter), (SEQ ID NO: 234), plant expression vectorplasmids, also digested with XbaI and SacI (Roche) and purified. pKGplasmid is based on the PCR Script backbone (GeneArt), with severalchanges in the polylinker site to facilitate cloning a gene of interestdownstream to a promoter and upstream to a terminator, suitable forexpression in plant cells. Moreover, the inserted gene, together withthe promoter and the terminator could be easily moved to a binaryvector.

The resulting pKG(NOSter) and pKG(35S+NOSter) harboring putative MonocotABST genes were introduced into E. coli DH5 competent cells byelectroporation, using a MicroPulser electroporator (Biorad), 0.2 cmcuvettes (Biorad) and EC-2 electroporation program (Biorad). The treatedcells were cultured in LB liquid medium at 37° C. for 1 hour, platedover LB agar supplemented with ampicillin (100 mg/L; Duchefa) andincubated at 37° C. for 16 hrs. Colonies that developed on the selectivemedium were analyzed by PCR using the primers of SEQ ID NO: 231 and SEQID NO: 232, which were designed to span the inserted sequence in the pKGplasmids. The resulting PCR products were separated on 1% agarose gelsand from the colonies having the DNA fragment of the predicted size, aplasmid was isolated using miniprep Plasmid Kit (Qiagen) and sequencedusing the ABI 377 sequencer (Amersham Biosciences Inc) in order toverify that the correct DNA sequences were properly introduced to the E.coli cells.

Positive pKG(NOSter) plasmids harboring putative Monocot ABST genes weredigested with the restriction enzymes HindIII and SalI (Roche), purifiedusing PCR Purification Kit (Qiagen, Germany), and then ligated (asdescribed above) with At6669 promoter sequence (set forth in SEQ ID NO:20) digested from pPI+At6669 plasmid with the same enzymes and purified.The resulting plasmids were introduced into E. coli DH5 competent cellsby electroporation, the treated cells were cultured in LB liquid mediumat 37° C. for 1 hr, subsequently plated over LB agar supplemented withampicillin (100 mg/L; Duchefa) and incubated at 37° C. for 16 hours.Colonies grown on the selective medium were analyzed by PCR using theprimers SEQ ID NO: 235 and SEQ ID NO: 232. Positive plasmids wereidentified isolated and sequenced as described above.

The plasmid pPI was constructed by inserting a synthetic poly-(A) signalsequence, originating from pGL3 basic plasmid vector (Promega, Acc NoU47295; by 4658-4811) into the HindIII restriction site of the binaryvector pBI101.3 (Clontech, Acc. No. U12640).

The At6669 promoter was isolated from Arabidopsis thaliana var Col0genomic DNA by PCR amplification using the primers set forth in SEQ IDNOs: 37 and 38. The PCR product is purified (Qiagen, Germany) anddigested with the restriction endonucleases HindIII and SalI (Roche).The resulting promoter sequence was introduced into the open binary pPIvector digested with the same enzymes, to produce pPI+At6669 plasmid.

Example 14 Generating Binary Vectors Comprising Putative Monocot ABSTGenes and Plant Promoters Operably Linked Thereto

Generating Binary Vectors Comprising the Cauliflower Mosaic Virus 35SPromoter:

The five pKG(35S+NOSter) constructs harboring putative Monocot ABSTgenes (SEQ ID Nos: 93, 94, 95, 96, 97 and 98) were digested with HindIIIand EcoRI (Roche) restriction endonucleases in order to exciseexpression cassettes and ligated to pPI plasmid digested with the sameendonucleases and purified (as described above). Altogether, five pPIconstructs were generated, each comprising putative Monocot ABST genehaving a sequence set forth in SEQ ID NOs: 93, 94, 95, 96, 97 and 98positioned downstream to the Cauliflower Mosaic Virus 35S promoter andupstream to the Nopaline Synthase (NOS) terminator, which was originatedfrom the digestion of pBI101.3 (Clontech, Acc. No. U12640), using therestriction sites SacI and EcoRI.

Generating Binary Vectors Comprising the At6669 Promoter:

The five pKG(At6669+NOSter) constructs harboring putative Monocot ABSTgenes downstream to At6669 promoter sequence (set forth in SEQ ID NO:20), and upstream to the Nopaline Synthase (NOS) terminator, weredigested with HindIII and EcoRI (Roche) in order to excise expressioncassettes and ligated into pPI plasmid which was digested with the samerestriction endonucleases and purified (as described above). Altogether,five pPI constructs were generated, each comprising the At6669 promoterpositioned upstream of a putative Monocot ABST gene having a sequenceset forth in SEQ ID NOs: 93, 94, 95, 96, 97 and 98.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents, patent applicationsand sequences identified by their accession numbers mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application or sequence identified bytheir accession number was specifically and individually indicated to beincorporated herein by reference. In addition, citation oridentification of any reference in this application shall not beconstrued as an admission that such reference is available as prior artto the present invention.

REFERENCES CITED

-   -   (Additional references are cited hereinabove)

-   1. World Wide Web (dot) fao (dot) org/ag/agl/agll/spush/degrad (dot)    htm.

-   2. World Wide Web (dot) fao (dot)    org/ag/agl/aglw/watermanagement/introduc (dot) stm

-   3. McCue K F, Hanson A D (1990). Drought and salt tolerance: towards    understanding and application. Trends Biotechnol 8: 358-362.

-   4. Flowers T J, Yeo Ar (1995). Breeding for salinity resistance in    crop plants: where next? Aust J Plant Physiol 22:875-884.

-   5. Nguyen B D, Brar D S, Bui B C, Nguyen T V, Pham L N, Nguyen H T    (2003). Identification and mapping of the QTL for aluminum tolerance    introgressed from the new source, ORYZA RUFIPOGON Griff., into    indica rice (Oryza sativa L.). Theor Appl Genet. 106:583-93.

-   6. Sanchez A C, Subudhi P K, Rosenow D T, Nguyen H T (2002). Mapping    QTLs associated with drought resistance in sorghum (Sorghum    bicolor L. Moench). Plant Mol Biol. 48:713-26.

-   7. Quesada V, Garcia-Martinez S, Piqueras P, Ponce M R, Micol J L    (2002). Genetic architecture of NaCl tolerance in Arabidopsis. Plant    Physiol. 130:951-963.

-   8. Apse M P, Blumwald E (2002). Engineering salt tolerance in    plants. Curr Opin Biotechnol. 13:146-150.

-   9. Rontein D, Basset G, Hanson A D (2002). Metabolic engineering of    osmoprotectant accumulation in plants. Metab Eng 4:49-56

-   10. Clough S J, Bent A F (1998). Floral dip: a simplified method for    Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant    J 16:735-43.

-   11. Desfeux C, Clough S J, Bent A F (2000). Female reproductive    tissues are the primary target of Agrobacterium-mediated    transformation by the Arabidopsis floral-dip method. Plant Physiol    123:895-904.

What is claimed is:
 1. A method of increasing abiotic stress tolerance,biomass and/or yield of a plant, comprising transforming a cell of theplant with a nucleic acid construct which comprises an exogenouspolynucleotide encoding a polypeptide at least 80% homologous to theamino acid sequence encoded by the polynucleotide selected from thegroup consisting of SEQ ID NOs: 1-18, 93-98 and 247-252, therebyincreasing the abiotic stress tolerance, biomass and/or yield of theplant.
 2. A method of producing a crop comprising growing a crop planttransformed with a nucleic acid construct which comprises an exogenouspolynucleotide encoding a polypeptide at least 80% homologous to theamino acid sequence encoded by the polynucleotide selected from thegroup consisting of SEQ ID NOs: 1-18, 93-98 and 247-252, wherein saidcrop plant is derived from plants selected for increased abiotic stresstolerance, increased biomass and/or increased yield as compared to awild type plant of the same species which is grown under the same growthconditions, and said crop plant having said increased abiotic stresstolerance, said increased biomass, and/or said increased yield, therebyproducing the crop.
 3. A method of selecting a plant for increasedabiotic stress tolerance, increased biomass and/or increased yield ascompared to a wild type plant of the same species which is grown underthe same growth conditions, the method comprising: (a) providing plantstransformed with a nucleic acid construct which comprises an exogenouspolynucleotide encoding a polypeptide comprising an amino acid sequenceat least 80% homologous to the amino acid sequence encoded by thepolynucleotide selected from the group consisting of SEQ ID NOs: 1-18,93-98 and 247-252, (b) selecting from said plants of step (a) a planthaving increased abiotic stress tolerance, increased biomass and/orincreased yield as compared to a wild type plant of the same specieswhich is grown under the same growth conditions, thereby selecting theplant having the increased abiotic stress tolerance, increased biomassand/or increased yield as compared to the wild type plant of the samespecies which is grown under the same growth conditions.
 4. The methodof claim 1, wherein said polypeptide is selected from the groupconsisting of SEQ ID NOs: 39-92 and 105-230.
 5. The method of claim 1,wherein said exogenous polynucleotide at least 80% identical to apolynucleotide selected from the group consisting of SEQ ID NOs: 1-18,93-98 and 247-252.
 6. The method of claim 1, wherein said exogenouspolynucleotide at least 90% identical to a polynucleotide selected fromthe group consisting of SEQ ID NOs: 1-18, 93-98 and 247-252.
 7. Themethod of claim 1, wherein said exogenous polynucleotide is selectedfrom the group consisting of SEQ ID NOs: 1-18, 93-98 and 247-252.
 8. Themethod of claim 1, wherein said nucleic acid construct comprising atleast one promoter capable of directing transcription of said exogenouspolynucleotide in said cell of the plant.
 9. The method of claim 1,further comprising growing the plant under the abiotic stress.
 10. Themethod of claim 1, wherein the plant is a dicotyledonous plant.
 11. Themethod of claim 1, wherein the plant is a monocotyledonous plant. 12.The method of claim 1, wherein said nucleic acid construct comprises aconstitutive promoter.
 13. The method of claim 1, wherein said nucleicacid construct comprises an inducible promoter or a tissue-specificpromoter.
 14. A nucleic acid construct comprising a polynucleotideencoding a polypeptide at least 80% homologous to the amino acidsequence encoded by the polynucleotide selected from the groupconsisting of SEQ ID NOs: 1-18, 93-98 and 247-252 and a heterologouspromoter capable of directing transcription of the polynucleotide in ahost cell.
 15. The nucleic acid construct of claim 14, wherein saidpolypeptide is selected from the group consisting of SEQ ID NOs: 39-92and 105-230.
 16. The nucleic acid construct of claim 14, wherein saidpolynucleotide at least 90% identical to a polynucleotide selected fromthe group consisting of SEQ ID NOs: 1-18, 93-98 and 247-252.
 17. Thenucleic acid construct of claim 14, wherein said polynucleotide isselected from the group consisting of SEQ ID NOs: 1-18, 93-98 and247-252.
 18. A plant cell comprising the nucleic acid construct of claim14.
 19. The plant cell of claim 18, wherein the plant cell forms part ofa plant.
 20. A transgenic plant comprising the nucleic acid construct ofclaim 14.